Probiotic Immunomodulatory Compositions

ABSTRACT

The invention discloses an immunomodulatory composition comprising non-culturable bacteria, a method of production and an article and an apparatus for its use and administration.

FIELD

The present disclosure is related to the field of immunomodulatorycompositions. In more detail, it relates to compositions obtainable byprocessing material derived from nature, to methods for theirmanufacturing and to their use.

BACKGROUND

Previously, immune system has been treated using cultivated probioticsthat are either single bacterial strains or combinations of rarecultivated strains. The beneficial effects of these products coverreduced probability of diarrhea and other gastrointestinal symptoms.Their use has been also studied in the prevention of otitis media and inthe treatment of alcoholic liver diseases (Sung et al. 2016),eradication of helicobacteria (McFarland et al. 2016), treatment ofdepression (Huang et al. 2016), maintenance of healthy vaginalmicrobiome during menopause (Muhleisen and Herbst-Kralovetz 2016) and inthe context of many other immune health related problems. Probiotics arealso recommended for pregnant women and for infants, as there isevidence that they reduce the probability of the upcoming child todevelop atopy. Probiotic-related treatments are also on the market, suchas Dirt shampoo where the active component is said to be a single,cultivated Mycobacterium species that has reduced mental disorders inrodents. Also parasitic worms and helminths have been experimented inimmunotherapy (Fumagalli et al. 2009; Schuijs et al. 2016; Matisz et al.2015).

In addition to probiotics, human vaginal fluids and human stool havebeen used to transfer microflora from human to human (Dominguez-Bello etal. 2016, Bakken 2009). The health effects of maternal vaginal fluids oninfants born in cesarean section are unclear, and stool transfer i.e.fecal bacteriotherapy is known to cure Clostridium difficile infection(Bakken 2009). Due to potential pathogen transfer, the donor of vaginalfluids is always infant's own mother. As there are concerns of pathogentransmission, a synthetic mixture of cultivated strains has beenproposed to avoid pathogenic microbes (Petrof et al. 2013). Fecaltransplantation has been proven effective for only few diseases and notfor diseases that are treated by modulating immune system.

Another, more traditional branch of treating immune system is the use ofchemicals, like immunosuppressive drugs. Prebiotics that modify theactivity of existing human microflora are available (Hutkins et al.2016, Schloss 2014). The reason for the desired effect of prebiotics isin chemical compounds, such as dietary fibers, not in themicrobiological content of prebiotics (Gibson, Roberfroid 1995). Naturalallergens are also used to treat allergies, e.g. birch pollen is used totreat pollinosis. The combination of several animal and hey allergens isalso marketed to prevent allergy. There are also speculativechemical-based treatments that are said to prevent or even cureallergies or immune system disorders.

The currently available treatments of immune system disorders and immunerelated diseases cover the use of chemicals (drugs, prebiotics andpurified allergens). In addition, cultivated microbial strains and humanexcretions have been used for this purpose, but there is only limitedevidence of their efficacy and their long-term use required forprophylaxis is not convenient for the subjects.

SUMMARY

In natural terrestrial environments and healthy human gut, a significantproportion—typically 40-99%—of bacteria are non-culturable. This resultsin enormous microbial diversity that is not properly utilized in knownproducts targeting immune system. The reasons are that non-culturablemicrobial communities are spatially and temporally heterogeneous, theyare not easily enriched, and that they often contain pathogenic species.In the current invention, the high microbial diversity is utilized inimmunomodulatory compositions that lack the spatial and temporalheterogeneity of natural environments and contain only a limited amountof or no potentially pathogenic species.

In contrast to previous attempts to provide immunomodulatorycompositions or microbiome transplantations comprising only a single orvery few species of culturable bacteria, fecal or vaginal microbes, thepresent invention uses natural, safe, and microbiologically very richmaterial. An advantage of the present invention is that a beneficialchange in the gut microbiome can be seen when using the presentimmunomodulatory composition. No such an effect has been obtained beforewhen orally administered probiotic bacteria were used. According to thefirst aspect is provided an immunomodulatory composition comprising amicrobial community comprising non-culturable bacteria, wherein

-   -   i. the microbial diversity of the immunomodulatory composition        is at least 3 at Shannon diversity index;    -   ii. the microbial richness of the immunomodulatory composition        is at least 130 operational taxonomic units; and/or    -   iii. the microbial abundance of the immunomodulatory composition        is at least 1000000 bacterial 16S copies g-1 ww; and/or    -   the abundance of pathogens in the immunomodulatory composition        is not higher than found in everyday living environment.

In an embodiment of the first aspect the immunomodulatory compositioncomprises material obtainable by processing material derived fromnature, wherein

-   -   a. material derived from nature comprises a microbial community        comprising non-culturable bacteria;    -   b. the microbial diversity of material derived from nature is at        least 3 at Shannon diversity index;    -   c. the immunomodulatory composition comprises a microbial        community comprising non-culturable bacteria; wherein    -   i. the microbial diversity of the immunomodulatory composition        is at least 3 at Shannon diversity index;    -   ii. the microbial richness of the immunomodulatory composition        is at least 130 operational taxonomic units; and/or    -   iii. the microbial abundance of the immunomodulatory composition        is at least 1000000 bacterial 16S copies g-1 ww; and    -   d. the abundance of pathogens in the immunomodulatory        composition is not higher than found in everyday living        environment.

The inventors surprisingly found that the immunomodulatory compositioncan be used to modulate microbial diversity, increase microbialdiversity, microbial abundance and microbial richness of a subjectliving in a modern society, preferably in urban conditions. The exposureis safe because pathogen levels can be controlled in the composition.This facilitates to reach similar or near similar microbial abundance(Example 1), diversity (Example 2) and richness (Example 1) as observedfor subjects that have remarkably low abundance of autoimmune diseasesand other immune system disorders, like allergies. Examples of theselow-risk subjects cover traditional hunter-gatherers (Schnorr et al.2014), children living on traditional dairy farms (Stein et al. 2016)and people living in societies where hygiene standards are not the sameas in present-day Western societies (Yatsunenko et al. 2012). Withoutbeing bound to any theory, living in modern urban environment causeschanges in the natural microbiome of the subject and home dust (Aleniuset al. 2009), which may lead to disorders of the immune system or weakenits efficiency. When the subject is exposed to the immunomodulatorycomposition, the microbial diversity, abundance and richness or at leastone of these factors is shifted closer to that observed among low-risksubjects, and therefore the immunomodulatory composition prevents orremedies the immune system related disorders and sicknesses, or improvesthe efficiency of the immune system. As evidenced by the examples, thepresent immunomodulatory composition causes increase in microbialabundance, richness, diversity and provides beneficial changes in theIL10-mediated immunoregulatory response (Example 3). An advantageobtainable with the immunomodulatory composition is that subjectsreceiving the immunomodulatory composition avoid risks of infection andother problems encountered by pathogens and pests. This is a strikingdifference with traditional hunter-gatherers and many nomads who alsoreceive a rich microbial exposure but who suffer from pathogens andpests.

The processing, which is carried out to obtain the immunomodulatorycomposition, is preferably such that the resulting composition retainssufficiently non-culturable bacteria or at least their active componentsthat are present in the material derived from nature, and that are ableto produce an immunomodulatory effect.

It has been shown that beneficial microbial changes can induce IL-10mediated immunoregulatory response for example in Crohn disease (Sokolet al. 2008; Rossi et al 2016). The immunomodulatory composition of thefirst aspect is able to induce a robust immunoregulatory IL-10 responsein white blood cells, whereas the proinflammatory IFN-gamma responseremains much weaker (Example 3). TGF-β is another cytokine withimmunoregulatory characteristics. A suitable dosage of theimmunomodulatory composition can be selected to induce immunoregulatoryresponse without inducing inflammation. Both intact (living) andinactivated composition are able to induce a beneficial immunoregulatoryresponse in white blood cells.

The immunomodulatory composition of the first aspect is fundamentallydifferent from existing immunomodulatory compositions at least for tworeasons: it comprises non-culturable microbial community that originatesin a diverse environment, and its level of pathogens is controlled. Inan embodiment the level of pathogens is controlled to a level not higherthan found in everyday living environment.

According to an aspect is provide a topical composition comprising theimmunomodulatory composition of the first aspect.

According to the second aspect is provided an immunomodulatorycomposition for use in regulating, maintaining and/or strengtheningimmune system and immunological regulation of a subject, the usecomprising exposing the subject to the immunomodulatory compositionaccording to the first aspect. In an embodiment such a use is anon-medical use.

According to another aspect is provided an immunomodulatory compositionfor medical use in regulating, maintaining and/or strengthening immunesystem and immunological regulation of a subject, the use comprisingexposing the subject to the immunomodulatory composition according tothe first aspect.

According to another aspect is provided an immunomodulatory compositionfor use in immunological regulation of a subject, the use comprisingexposing the subject to the immunomodulatory composition according tothe first aspect. In an embodiment such a use is a non-medical use.

According to another aspect is provided an immunomodulatory compositionfor medical use in immunological regulation of a subject, the usecomprising exposing the subject to the immunomodulatory compositionaccording to the first aspect.

In another aspect is provided an immunomodulatory composition for use inmaintaining and strengthening immune system of a subject, the usecomprising exposing the subject to the immunomodulatory compositionaccording to the first aspect. In an embodiment such a use is anon-medical use.

In another aspect is provided an immunomodulatory composition formedical use in maintaining and strengthening immune system of a subject,the use comprising exposing the subject to the immunomodulatorycomposition according to the first aspect.

In another aspect is provided immunomodulatory composition of thepreceding aspects is for medical treatment of allergy. In an embodimentthe immunomodulatory composition is an IL-10 or TGF-β mediatedimmunomodulatory composition.

According to another aspect is provided an immunomodulatory compositioncomprising material derived from nature which contains non-culturablemicrobial community. In an embodiment the material comprises at leastone of industrial, mining, agricultural, silvicultural, aquacultural,water purification, water filtration or peat production materials,including byproducts that are currently being used for other purposeswithout knowing, understanding an utilizing the immunomodulatoryproperties of the materials. The authors surprisingly found that theseexisting materials may be suitable for immunomodulatory compositions,provided that pathogen levels are controlled.

In another aspect is provided the immunomodulatory composition fornon-medical regulating, maintaining and strengthening of immune system,wherein the subject is a human subject having a disorder of immunity ora human subject living in an urban environment wherein microbialdiversity, richness and/or abundance is lower than in the materialderived from nature.

According to the third aspect is provided a method for manufacturing animmunomodulatory composition according to the first aspect, the methodcomprising:

-   -   a. Providing material derived from nature to provide raw        material;    -   b. Grinding raw material to obtain particulate or powdered        material;    -   c. Homogenizing spatial variation of microbial community of the        powdered material preferably by sieving and mixing;    -   d. Optionally drying material obtained in b-c to provide dried        material wherein the number of living or metabolically active        cells of pathogenic species or microbial metabolism decreases;    -   e. Optionally adding water, oil, carrier or solvent to material        obtained in b-d to provide lotion, gel, cream or liquefied        material; and    -   f. Optionally sterilizing material provided in b-d to provide        sterilized material.

According to a fourth aspect is provided a method for manufacturing animmunomodulatory composition according to the first aspect, the methodcomprising

-   -   a. Providing material derived from nature to provide raw        material;    -   b. Extracting the raw material to provide an extract;    -   c. Optionally evaporating the extract to provide gaseous        material; and    -   d. Optionally condensing the evaporated extract.    -   e. Optionally sterilizing material provided in b-d to provide        sterilized material.

According to a fifth aspect is provided a method for manufacturing theimmunomodulatory composition of the first aspect, the method comprising:

-   -   a. Providing material derived from nature to provide raw        material    -   b. Grinding the raw material to obtain particulate raw material;    -   c. Sieving the particulate raw material to provide sieved raw        material;    -   d. Extracting the product obtained in step b., or c., to provide        an extract;    -   e. Optionally lyophilizing the extract; and    -   f. Optionally sterilizing material provided in d or to provide        sterilized material.

According to another aspect is provided a method for manufacturing animmunomodulatory composition according to the first aspect comprisingmaterial derived from nature, the method comprising:

-   -   a. Providing material derived from nature to provide raw        material;    -   b. Optionally grinding raw material to obtain particulate or        powdered material;    -   c. Optionally sieving material derived from nature or the        particulate or powdered material to provide sieved material;    -   d. Optionally oven-drying raw material or material obtained in        b-c to provide oven-dried material wherein the microbial        community composition or its metabolism changes in oven-drying;    -   e. Optionally moisturizing raw material or material obtained in        b-d to provide lotion-like or liquefied material;    -   f. Optionally lyophilizing raw material or material provided in        b-e to provide lyophilized material;    -   g. Optionally sterilizing raw material or material provided in        b-f to provide sterilized material;    -   h. Optionally evaporating material derived from nature or        material provided in b-g to provide evaporated material; wherein    -   the method comprises at least step a and any combination of        steps b-h performed successively.

According to another aspect is provided a method for manufacturing animmunomodulatory composition according to the first aspect comprising

-   -   a. Providing material derived from nature to provide raw        material;    -   b. Optionally grinding the raw material to obtain particulate or        powdered material;    -   c. Optionally sieving the particulate or powdered raw material        to provide sieved material;    -   d. Optionally oven-drying the raw material to provide oven-dried        material;    -   e. Optionally moisturizing the raw material to provide        lotion-like or liquefied material;    -   f. Optionally lyophilizing the raw material to provide        lyophilized material;    -   g. Optionally sterilizing the raw material to provide sterilized        material;    -   h. Optionally evaporating the raw material to provide evaporated        material;    -   i. Extracting the product obtained in step a., b., c., d., e.,        f., or g. to provide an extract;    -   j. Optionally lyophilizing the extract to provide the        lyophilized extract;    -   k. Optionally oven-drying the extract to provide oven-dried        extract;    -   l. Optionally evaporating the extract to provide gaseous        material;    -   m. condensing the evaporated extract obtained in step l. to        provide condensed material;    -   n. composting the raw material to provide composted material;        and    -   wherein the method comprises at least step a. and any        combination of steps b., c., d., e., f., g., h., i., j., k., l.,        m., and n.

According to the fifth aspect is provided an article for releasing theimmunomodulatory composition of the first aspect comprising acompartment for the composition.

According to the sixth aspect is provided an apparatus for administeringimmunomodulatory composition of the first aspect, wherein the apparatusis configured to receive a replaceable unit dose of the immunomodulatorycomposition of the first aspect.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 discloses a box-plot showing the abundance as the number ofbacterial 16S copies in hands before and after an exposure to differentimmunomodulatory compositions comprising non-culturable microbialcommunity. The compositions are different composted and sievedagricultural byproducts and other sieved materials derived from nature.Thick line is the median, the boxes show the lower and upper hinges andwhiskers show the most extreme data points. If the most extreme datapoint is at least 1.5 times the interquartile range of the box, it isshown as a circle. As FIG. 1 shows, the immunomodulatory composition ofthe first aspect comprising non-culturable microbial community increasesmicrobial abundance on human skin.

FIG. 2A-B discloses box-plots showing bacterial richness before andafter an exposure of hands to different immunomodulatory compositionscomprising non-culturable microbial community. The compositions aredifferent composted and sieved agricultural byproducts and other sievedmaterials derived from nature. Thick line is the median, the boxes showthe lower and upper hinges and whiskers show the most extreme datapoints. If the most extreme data point is at least 1.5 times theinterquartile range of the box, it is shown as a circle. P-values andstatistics (W) are based on Wilcoxon signed rank test. A) Number of OTUsand number of taxa at different taxonomic levels. B) Number of OTUswithin five dominant phyla (Phyl.), number of OTUs for unclassifiedbacteria at phylum level (Phyl. unclassified) and number of OTUs withinthree classes (Alpha-, Beta- and Gammaproteobacteria). As FIG. 2 shows,the immunomodulatory composition comprising non-culturable microbialcommunity increases microbial richness on human skin, and it alsoincreases the richness within major bacterial phyla, classes andunclassified bacteria on human skin.

FIG. 3A-3B discloses box-plots showing bacterial diversity before andafter an exposure of hands to different immunomodulatory compositionscomprising non-culturable microbial community. The compositions aredifferent composted and sieved agricultural byproducts and other sievedmaterials derived from nature. Thick line is the median, the boxes showthe lower and upper hinges and whiskers show the most extreme datapoints. If the most extreme data point is at least 1.5 times theinterquartile range of the box, it is shown as a circle. P-values andstatistics (W) are based on Wilcoxon signed rank test. A) Shannondiversity index based on all OTUs and taxa at different taxonomiclevels. B) Shannon diversity index based on OTUs within five dominantphyla (Phyl.), number of OTUs for unclassified bacteria at phylum level(Phyl. unclassified) and number of OTUs within three classes (Alpha-,Beta- and Gammaproteobacteria). As FIG. 3 shows, the immunomodulatorycomposition comprising non-culturable microbial community increasesmicrobial diversity on human skin, and it also increases the diversitywithin major bacterial phyla, classes and unclassified bacteria on humanskin.

FIG. 4. The bacterial ecosystem diversity i.e. community composition offecal samples in two groups of volunteers. The case group exposed theirhands to an immunomodulatory composition three times a day for twoweeks. The control group did not receive the immunomodulatorycomposition. Dissimilarity in community composition between groups wasplotted using Non-Metric Multidimensional Scaling (NMDS) withBray-Curtis dissimilarity data (details in example 2). Table 2 hascalculations used in revealing statistically significant ecosystemdiversity changes (Fisher's exact test, P-Value 0.029) between exposedand non-exposed subjects. Together with Table 2, the technical effect isthat the external use of the immunomodulatory composition according tothe current invention can increase species change and diversity in stoolmicrobial community.

FIG. 5A-5B. Stimulation of the peripheral blood mononuclear cells(PBMCs) with the extracted, freeze-dried and resuspendedimmunomodulatory composition that is called as soil mixture extract inFIG. 5A-5B. PBMCs were stimulated either with resuspended i.e. original,heat inactivated or filtered immunomodulatory composition for 24 and 48hours. Stimulation by anti-CD3/CD28 was used as a positive control inthe stimulation experiment. Panel A shows the IL-10 expression in PBMCsand panel B shows the IFN-gamma (i.e. IFNg) expression compared topositive anti-CD3/28 control. The technical effect is that theimmunomodulatory composition according to the current invention caninduce predominantly immunoregulatory IL-10-type responses while it doesnot induce proinflammatory IFN-gamma -type responses. In addition,killing the bacteria by heat-treatment did not abolish IL-10 responseswhile elimination of bacteria by filtration through 0.45 μm filterdecreased IL-10 responses considerably. This indicates that thefavorable immunoregulatory effect is dependent of the presence ofbacteria in the immunomodulatory composition.

FIG. 6. Microbial abundance as indicated by number of 16 S sequences inthe sieved immunomodulatory composition described in example 2, naturalforest soils described in examples 4 and 7, freeze driedimmunomodulatory composition described in example 3, commonly usedmineral soil materials from aggregate producer described in examples 4and 7, and mineral soils collected at urban daycare yards described inexample 7. For microbial diversity, richness, pathogen levels andsampling details, see examples 2-7. Note that the values in Mineralsoil—Aggregate producer are very low, and that those in Mineralsoil—Daycare yard are lower than in natural forests and immunomodulatorycompositions. The technical effect is that immunomodulatory compositionsaccording to the current invention are suitable for modifying microbialcommunities of a subject.

FIG. 7. Infant hat with tied top (left) and the same infant hat insideout (right). Note the pocket for the immunomodulatory packet. Thetechnical effect is that forehead skin becomes in contact with smallparticles of immunomodulatory composition. This will in turn activateimmunoregulatory mechanisms i.e. lead to a technical effect. Thistechnical effect can partially also be produced by inhaling theimmunomodulatory components while using the product.

FIG. 8. Sketch of infant hat (not to scale) shown in FIG. 7.

FIG. 9. Building block (left) and interior of building block (right). Ininterior, two packets of immunomodulatory composition are visible.Blocks enable the immunomodulatory components of the immunomodulatorymaterial to scatter on and around the children while using the product.This will in turn activate immunoregulatory mechanisms i.e. lead to atechnical effect. This technical effect can partially also be producedby inhaling the immunomodulatory components while using the product.

FIG. 10. Infant security blanket. Packet of immunomodulatory compositionis within the top that is tied with a ribbon into a bundle. The infantsecurity blankets have similar technical effects as the other products:the immunomodulatory component of the material passes through the fabriclayers and reaches the skin activating immunoregulatory mechanisms. Aswith the other products, the technical effect can partially be caused byinhaling the immunomodulatory components while using the product.

FIG. 11. Sketch of infant security blanket.

FIG. 12 Pillow case inside out (left) and duvet cover inside out(right). The technical effect of stimulating the immunoregulatory systemhere is analogous to the other products. The immunomodulatory componentof the immunomodulatory composition passes through the layers of fabricand reaches the skin activating immunoregulatory mechanisms. Similartechnical effect can, in part, also be triggered by the inhalation ofthe immunomodulatory components of the material while using the product.

FIG. 13 shows the effect of a packet containing an immunomodulatorycomposition on skin microbial community. Skin swab samples before andafter the use of packets filled with the immunomodulatory compositionhave different bacterial communities. Also the bacterial communitycomposition of material derived from nature, i.e., non-processedSphagnum moss is shown. For details of manufacturing theimmunomodulatory composition, see Example 9. The ordination method isprincipal coordinate analysis. For abundance data, the varianceexplained by the first and second axes are 44.5% and 27.6%,respectively. For presence-absence data, the variance explained by thefirst and second axes are 37.7% and 18.0%, respectively. Figuresvisualize the technical effect how the use of the packets withimmunomodulatory composition shifted the bacterial community compositionon skin; changes in richness and diversity are shown in Table 10.

FIG. 14 is a flow diagram showing alternative processing pathways tomanufacture the immunomodulatory composition from the material derivedfrom nature as a raw material. Star denotes a step in whichimmunomodulatory composition can be recovered.

SEQUENCE LISTINGS SEQ ID NO 1: oligonucleotide pE SEQ ID NO 2:oligonucleotide pF SEQ ID NO 3: oligonucleotide 505F SEQ ID NO 4:oligonucleotide 806R SEQ ID NO 5: oligonucleotide pA_Illumin_FPSEQ ID NO 6: oligonucleotide pD’_Illumin_RPSEQ ID NO 7: oligonucleotide pA 1: AGAGTTTGATCMTGGCTCAGSEQ ID NO 8: oligonucleotide pA 2: TAGAGAGTTTGATCMTGGCTCAGSEQ ID NO 9: oligonucleotide pA 3: CTCTAGAGTTTGATCMTGGCTCAGSEQ ID NO 10: oligonucleotide pD’ 1: GTATTACCGCGGCTGCTGSEQ ID NO 11: oligonucleotide pD’ 2: CGTATTACCGCGGCTGCTGSEQ ID NO 12: oligonucleotide pD’ 3: TAGTATTACCGCGGCTGCTGSEQ ID NO 13: oligonucleotide pD’ 2:ATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTSEQ ID NO 14: oligonucleotide pD’ 3: GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT

DETAILED DESCRIPTION

The present invention is based on the new finding that immunomodulatorycomposition based on the material derived from nature can be used toprevent or cure immune system associated disorders and diseases or toenhance the efficacy or to maintain the health of the immune system.

Methods to Determine Threshold Values

The parameters characterizing the present invention were determined asexplained below. As is understood in the art, the skilled person may inspecial situation select an alternative or supplementary method fordetermining a parameter. Depending on the selected assay conditions,subsampling protocol, sampling depth, extraction method or Illumina kitor any other technical detail of the molecular biological orcomputational analysis may therefore provide different parameterscompared to what is obtained in the characterizing parameters of thecurrent invention. A change in a detail of the analysis may affect athreshold value. The examples below provide alternative protocols ofdetermining threshold values. However, the parameters used in claims canbe determined by the skilled person by following the teaching below.

Sample Preparation for MiSeq Sequencing

Samples are stored in deep freezer (<−70° C.) before DNA extraction.Material wet weight used in DNA extraction is 0.25 g. Total DNA isextracted from samples using PowerSoil® DNA Isolation Kit (MoBioLaboratories, Inc., Carlsbad, Calif., USA) according to themanufacturer's standard protocol and DNA is checked with agarose gel(1.5%) electrophoresis (120 V, 30 min). The total DNA concentration ismeasured with Quant-iT™ PicoGreen® dsDNA reagent kit (Thermo scientific,MA, USA). The DNA concentration is adjusted to 0.35-0.4 ng/μl in eachsample. DNA is analyzed for bacterial (16S) communities using a two-stepPCR approach to avoid a 3′-end amplification bias resulting from thesample-specific DNA tags (Berry et al. 2011). The V4 region within the16S ribosomal RNA (rRNA) gene is amplified by primary PCR using 505F and806R primers (Caporaso et al. 2012). Primary PCR is carried out in areaction mixture (reaction volume 50 μl) consisting of 1 μl each of 10mM deoxynucleoside triphosphates (dNTPs; Thermo scientific, MA, USA) 5μl forward primer 505F (10 μM; 5′-GTGCCAGCMGCCGCGGTAA-3′) and 5 μlreverse primer 806R (10 μM; 5′-GGACTACHVGGGTWTCTAAT-3′), 0.5 μl 2 U/μlPhusion Green Hot Start II High-Fidelity DNA polymerase (Thermoscientific, MA, USA), 10 μl 5× Green HF PCR buffer (F-537), 5 μltemplate DNA and 23.5 μl sterile water. The PCR reaction is run in athermocycler as follows: initial denaturation at 98° C. for 5 min,followed by 25 cycles with denaturation at 94° C. for 1 min, annealingfor 10 sec at 50° C. and extension for 1 min at 72° C., and then a finalextension at 72° C. for 10 min. A negative control (sterile water) isused to detect and later adjust data for any possible contamination. DNAwas detected with agarose gel (1.5%) electrophoresis (120 V, 1 h). ThePCR products are purified using Agencourt AMPure XP solution (BeckmanCoulter Ins.) to reduce carryover of primary PCR primers. The cleanedamplicons are diluted 1:5. Cleaned and diluted primary PCR products aretargeted in the secondary PCR (TagPCR). Reaction mixture to the TagPCRis equal as above except reverse primer include a 12 bp uniqueMultiplexing Identifier tag (MID-806R). Amplification program is thesame as above except that only ten cycles are used. TagPCR products aredetected on agarose gel (1.5%) electrophoresis (120 V, 1 h), purifiedwith Agencourt AMPure, pooled and the DNA concentration is measured withPicoGreen. The sequencing is performed using Illumina MiSeq platformwith a 2×300 bp version 3 kit sequencing kit according to manufacturer'sprotocol. The GeneRead DNA Library I Core Kit (Qiagen, catalog #180432)is used to ligate Illumina's TruSeq adapters to amplicons.

Sequence Processing

The paired sequences contained in reverse and forward fastq files arealigned into a contig. The resulted library is trimmed and screened toremove any mismatches with primer or DNA-tag sequences, ambiguous basesand homopolymers larger than 8 bp long. Sequences are aligned usingMothur version of SILVA bacterial reference sequences (version 102;Pruesse et al. 2007) and the sequences, which are not aligned to areference alignment of the correct sequencing region are removed. Uniquesequences and their frequency in each sample are identified, and then,almost identical sequences (>99% similar) are preclustered to minimizesequencing errors (Huse et al. 2010) and screened for chimeras (UCHIME,Edgar et al. 2011) using the abundant sequences as a reference. Thechimeric sequences are removed. The sequences are classified usingMothur version of Bayesian classifier (Wang et al. 2007) with the RDPtraining set version 9 (Cole et al 2009). Sequences that are classifiedas Mitochondria, Chloroplast, Archaea, Eukaryota or unknown are removed.Operational taxonomic units (OTUs) are assigned at 97% identity. RareOTUs that are represented with 10 or fewer sequences in the whole dataare removed. For each OTU the number of sequences detected in control issubtracted from the samples and negative values are changed to zeros.Finally, all the samples are rarefied to 2000 sequences. If the sampleincludes less than 2000 sequences, it is not considered further.

qPCR Method

Quantitative PCRs are carried out with the Light Cycler 96 Quantitativereal-time PCR machine (MJ Research, MA, USA). The forward primer is pE5′-AAA CTC AAA GGA ATT GAC GG-3′ (SEQ ID NO:1) and the reverse primer pF5′-ACG AGC TGA CGA CAG CCA TG-3′ (SEQ ID NO: 2) (Öqvist et al. 2008).All samples are run in triplicates in 20 μl reactions containing 10 μl2× PowerUp SYBR Green Master Mix (Thermo scientific, MA, USA), 0.2 μl 20mg/ml BSA, 0.5 μl of each primer (10 μM), and the sample template. Astandard curve is included in every run to allow quantitation of thenumber of bacterial 16S copies present in the original sample. The q-PCRrun is as follows: initial denaturation at 95° C. min, followed by 40cycles of denaturation at 95° C. for 10 s, annealing for 20 sec at 53°C. and extension for 30 s at 72° C.

Terminology

As used herein, the term “comprising” includes the broader open meaningsof “including”, “containing”, and “comprehending”, as well as thenarrower closed meanings of the expressions “consisting of” and“consisting only of”.

The term immunomodulatory is multifaceted in the context of thisapplication and it includes at least the following aspects: In oneaspect it refers to the stimulation of the immune response to theforeign and harmful materials such as pathogens. It also refers to theoverall enhancement of the health, function and potency of the immunesystem. It also refers to the training of the immune system to responseappropriate and healthy ways for different stimuluses and to avoidunhealthy pathological responses. It also covers immunoregulatory andimmunostimulatory aspects to tune the immune response in a healthy wayand to avoid unhealthy responses. In one aspect it also refers to themaintaining of the healthy status of immune system and immune responseor to slowing down the decline of the immune system and immune responsefor example in the cases of immune system affecting diseases and aging.

The term material derived from nature refers to soil material, plantmaterial, algal, fungal or Protozoan material or non-human animalmaterial, which comprises non-culturable bacteria, and wherein theamount of pathogenic bacteria and viruses drop during processing belowor to the level that the subjects are exposed to in their everyday life.Alternatively, the level of pathogenic bacteria and viruses arecontinuously below or at the level that the subjects are exposed to intheir everyday life.

The abundance of pathogens found in everyday living environment mayvary. Example 7 provide example of abundances found in exemplaryeveryday living environments.

The term material derived from nature refers to any natural raw materialor byproduct that comprises at least 40 different OTUs at at least 97%similarity level.

In an embodiment the microbial diversity of the immunomodulatorycomposition is at least 3 at Shannon diversity index at at least 97%similarity level; the microbial richness of the immunomodulatorycomposition is at least 130 operational taxonomic units at at least 97%similarity level; and/or the microbial abundance of the immunomodulatorycomposition is at least 1000000 bacterial 16S copies g-1 ww. In anotherembodiment the level of pathogens in the immunomodulatory composition isnot higher or not significantly higher than found in everyday livingenvironment.

In an embodiment the material derived from nature comprises agriculturalmaterial streams, such as agricultural plant waste or solid plant waste,milling waste, oil extraction waste, roots, peels, seeds, seed pods,leaves, litter, inflorescence, cones or membranes, or animal byproductssuch as animal dung, sludge, internal organs, skin, eggshells, shells orcarcasses or their parts.

In an embodiment the material derived from nature comprisessilvicultural material streams, such as bark, saw dust, wood chip,shaved wood chip, pulp, needles, leaves, branches, roots, tree litterand other products and byproducts that contain microbial communitycomprising non-culturable bacteria;

In an embodiment the material derived from nature comprises aquaculturalmaterial streams, including byproducts, such as cultivated aquaticanimals and their carcasses or body parts, algae, aquatic plants,plankton and debris from the organisms mentioned herein that containmicrobial community comprising non-culturable bacteria;

In an embodiment the material derived from nature comprises waterpurification streams, including byproducts, such as material comprisingnon-culturable bacteria originating in water filtration systems.

In an embodiment the material derived from nature comprises materialstreams in production and mining industry, including byproducts, such aspeat, plant debris, moss and microbial communities flourishingunintentionally during biomining.

In an embodiment the material derived from nature comprises a selectedcombination of composted and sieved soil and plant-based ingredientsincluding 861 OTUs (≥97% similarity) from 19 phyla based on 16S rRNAsequencing. In an embodiment the two most abundant phyla areBacteroidetes and Proteobacteria.

In an embodiment the material derived from nature does not comprisepeat.

In another embodiment the material derived from nature does not compriseanimal dung or human stool. Such an embodiment is advantageous becauseof easier control of pathogens and lower initial level of fast growingbacteria in the material.

The term material derived from nature also refers to industrial productsthat exist, comprise community in claim 1a, comprise at least 40different OTUs at at least 97% similarity level and are produced andused only for other purposes outside this patent, regardless of theirmicrobiological properties.

The term operational taxonomic unit (shortened as OTU) refers toclusters of 16S or 18S small subunit rRNA gene similarity and it is usedas an approximation of microbial taxa (Schmidt et al. 2014). In theclaims of the current patent application, we specifically define thatthe term OTU is determined using variable region 4 (i.e. V4) inbacterial 16S rRNA gene and 97% similarity. OTUs are clustered usingnearest neighbor algorithm in Mothur where each of the sequences withinan OTU are at most 3% distant from the most similar sequence in the OTU.(https://mothur.org/wiki/Cluster, accessed 5 Dec. 2016) This facilitatescomparison of the claims presented herein to any other potentialapplications in the future. It should be noted that when analyzing theeffect of the immunomodulatory composition, OTUs can be defined usingother variable regions, such as variable regions 1-3 that were used inexample 1.

The term non-culturable bacteria refers to bacteria that are viable andmetabolically or physiologically active, but not culturable. Thebacterial cells that form a colony on specific nutrient media aredefined as culturable bacteria. So the bacteria that are metabolicallyor physiologically active but cannot be cultured on specific media arethe viable but non-culturable bacteria (Fakruddin et al., 2013).Regarding this by using modern molecular recognition tools such as 16SrRNA sequencing the number of divisions of bacteria has grown from 11 toat least 85 after year 1989, and the majority of these divisions have nocultured representatives (Stewart, 2012).

The term abundance refers to the total number of 16S or 18S gene copiesper g wet weight.

The term wet weight (shortened as ww) refers to sample weight whereinwater is included. In an embodiment water content of a wet weight sampleis 0.1-80%, such as 50%.

Terms subsampling rarefying and rarefaction (Hughes and Hellmann 2005)refer to a procedure where samples are scaled down to an equal number ofsequences by randomly picking the same number of sequences from eachsample.

The terms Shannon diversity index or Shannon index (Shannon, 1948) referto a commonly used measure of diversity, which measures the uncertaintyto correctly predict the taxa of the next individual collected (Krebs2001). The index value increases when (1) the number of taxa increasesand (2) the more evenly the abundances of sequences are distributedamong taxa. The index can be calculated using different logarithmicbases but here the index is calculated using natural logarithm and thusthe equation is as follows:

$H = {- {\sum\limits_{i = 1}^{s}{p_{i}\ln p_{i}}}}$

where p_(i) is the proportional abundance of a taxon i.

The term richness refers to the number of OTUs in a sample. Preferably,richness refers to OTU similarity of 97%. Richness can also refer to thenumber of taxa (e.g. genera, classes, phyla) in a sample.

In an embodiment the immunomodulatory composition contains a diversityof metabolically almost inactive microbes that are rare in urban spaceand usually abundant in natural environments. Without being bound to anytheory, they induce an immunoregulatory response in a subject exposed tothem.

The term sterilizing or inactivation refers to irradiating, autoclaving,boiling, steaming and other methods suitable for inactivating or killingliving microbes and pathogens or destructing their outer membranes.

In another embodiment the immunomodulatory composition comprises aqueousextract obtained by extracting material derived from nature by anaqueous solvent.

Pathogens

In an embodiment the material derived from nature is essentially freefrom pathogens.

In an embodiment the immunomodulatory composition is free frompathogens.

In an embodiment the level of pathogens in the immunomodulatorycomposition is below a level of 50 sequences per 0.25 g ww sample inbacterial genera that were classified as potentially pathogenic byTaylor et al. (2001). These comprise of the following genera:Acinetobacter, Actinomyces, Aerococcus, Aeromonas, Arcobacter, Bacillus,Bacteroides, Bifidobacterium, Brevibacillus, Brevundimonas,Chryseobacterium, Corynebacterium, Fibrobacter, Finegoldia, Gemella,Lactobacillus, Legionella, Leptotrichia, Moraxella, Mycobacterium,Myroides, Neisseria, Nocardia, Paenibacillus, Prevotella, Pseudomonas,Pseudonocardia, Psychrobacter, Rhodococcus, Rickettsia,Saccharomonospora, Sphingomonas, Stenotrophomonas, Streptococcus, andTreponema. As evidenced by example 7, the total number of sequences inall the potentially pathogenic bacterial genera listed by Taylor et al.(2001) is lower in the immunomodulatory composition than what is foundin everyday living environment of urban children (sandpits on daycareyards). In an embodiment the pathogen refers to a strain of above generahaving pathogenic characteristics.

In an embodiment the level of pathogens in the immunomodulatorycomposition is below a level of 100, 200, 300, 400 or 500 sequences per0.25 g ww sample in bacterial genera that were classified as potentiallypathogenic by Taylor et al. (2001).

In an embodiment the sample is subsampled.

In an embodiment, the level of certain pathogens is determined usingcultivation in laboratory conditions. As evidenced by example 6, themanufacturing methods of the immunomodulatory composition guarantee thatit lacks pathogenic Pseudomonas aeruginosa. In yet another embodiment ofthe first aspect the abundance of Escherichia coli and Pseudomonasauriginosa in the immunomodulatory composition is 0, as evidenced inexamples 6 and 7.

In an embodiment, the level of pathogens is determined using a Q-PCRbased method. As evidenced by example 5, the manufacturing methods ofthe immunomodulatory composition guarantee that it lacks the followingviral and protozoan pathogens: enterovirus, rhinovirus, rotavirus,norovirus, Giardia and Cryptosporidium.

In an embodiment, the level of pathogens can be determined using e.g.Illumina sequencing of bacterial community. As evidenced by example 7,the manufacturing methods of the immunomodulatory composition guaranteethat it lacks the following bacterial pathogenic genera: Bacillus,Escherichia, Neisseria and Nocardia.

Taxa

In an embodiment non-culturable bacteria that are present in theimmunomodulatory composition are selected from the phyla Actinobacteria,Acidobacteria, Bacteroidetes, Firmicutes, Proteobacteria, or acombination thereof. Within Proteobacteria, the selection can be madefrom Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria. Inthe embodiment presented in example 3, non-culturable bacteria that arepresent in the immunomodulatory composition are selected from phylaAcidobacteria, Actinobacteria, Armatimonadetes, Bacteroidetes, BRC1,Chlamydiae, Chlorobi, Chloroflexi, Deinococcus-Thermus, Firmicutes,Gemmatimonadetes, Nitrospira, OD1, OP11, Planctomycetes, Proteobacteria,TM7, and Verrucomicrobia; classes Acidobacteria_Gp1, Acidobacteria_Gp10,Acidobacteria_Gp16, Acidobacteria_Gp17, Acidobacteria_Gp2,Acidobacteria_Gp21, Acidobacteria_Gp22, Acidobacteria_Gp3,Acidobacteria_Gp4, Acidobacteria_Gp6, Acidobacteria_Gp7, Actinobacteria,Alphaproteobacteria, Anaerolineae,Armatimonadetes_gp2_class_incertae_sedis,Armatimonadetes_gp5_class_incertae_sedis, Armatimonadia, Bacilli,Bacteroidetes_incertae_sedis_class_incertae_sedis, Bacteroidia,Betaproteobacteria, BRC1_class_incertae_sedis, Caldilineae, Chlamydiae,Clostridia, Deinococci, Deltaproteobacteria, Flavobacteria,Gammaproteobacteria, Gemmatimonadetes, Ignavibacteria, Nitrospira,OD1_class_incertae_sedis, OP11_class_incertae_sedis, Opitutae,Planctomycetacia, Spartobacteria, Sphingobacteria, Subdivision3,Thermomicrobia, TM7_class_incertae_sedis, and Verrucomicrobiae; ordersAcidimicrobiales, Acidobacteria_Gp1_order_incertae_sedis,Acidobacteria_Gp10_order_incertae_sedis,Acidobacteria_Gp16_order_incertae_sedis,Acidobacteria_Gp17_order_incertae_sedis,Acidobacteria_Gp2_order_incertae_sedis,Acidobacteria_Gp21_order_incertae_sedis,Acidobacteria_Gp22_order_incertae_sedis,Acidobacteria_Gp3_order_incertae_sedis,Acidobacteria_Gp4_order_incertae_sedis,Acidobacteria_Gp6_order_incertae_sedis,Acidobacteria_Gp7_order_incertae_sedis, Actinomycetales,Alphaproteobacteria_order_incertae_sedis, Alteromonadales,Anaerolineales, Armatimonadales,Armatimonadetes_gp2_order_incetae_sedis,Armatimonadetes_gp5_order_incetae_sedis, Bacillales, Bacteroidales,Bacteroidetes_incertae_sedis_order_incertae_sedis, Bdellovibrionales,BRC1_order_incertae_sedis, Caldilineales, Caulobacterales, Chlamydiales,Clostridiales, Deinococcales, Deltaproteobacteria_order_incertae_sedis,Desulfuromonadales, Flavobacteriales,Gammaproteobacteria_order_incertae_sedis, Gemmatimonadales,Hydrogenophilales, Ignavibacteriales, Lactobacillales, Legionellales,Methylococcales, Myxococcales, Nitrosomonadales, Nitrospirales,OD1_order_incertae_sedis, OP11_order_incertae_sedis, Opitutales,Planctomycetales, Pseudomonadales, Puniceicoccales, Rhizobiales,Rhodospirillales, Rubrobacterales, Solirubrobacterales,Spartobacteria_order_incertae_sedis, Sphaerobacterales,Sphingobacteriales, Subdivision3_order_incertae_sedis,TM7_order_incertae_sedis, Verrucomicrobiales, and Xanthomonadales;families Acetobacteraceae, Acidimicrobineae_incertae_sedis,Acidobacteria_Gp1_family_incertae_sedis,Acidobacteria_Gp10_family_incertae_sedis,Acidobacteria_Gp16_family_incertae_sedis,Acidobacteria_Gp17_family_incertae_sedis,Acidobacteria_Gp2_family_incertae_sedis,Acidobacteria_Gp21_family_incertae_sedis,Acidobacteria_Gp22_family_incertae_sedis,Acidobacteria_Gp3_family_incertae_sedis,Acidobacteria_Gp4_family_incertae_sedis,Acidobacteria_Gp6_family_incertae_sedis,Acidobacteria_Gp7_family_incertae_sedis, Actinospicaceae,Alicyclobacillaceae, Alphaproteobacteria_family_incertae_sedis,Alteromonadaceae, Anaerolineaceae, Armatimonadaceae,Armatimonadetes_gp2_family_incetae_sedis,Armatimonadetes_gp5_family_incetae_sedis, Bacteriovoracaceae,Bacteroidetes_incertae_sedis_family_incertae_sedis, Bdellovibrionaceae,Bradyrhizobiaceae, BRC1_family_incertae_sedis, Caldilineaceae,Carnobacteriaceae, Caulobacteraceae, Clostridiaceae_1,Clostridiales_Incertae_Sedis_XVIII, Conexibacteraceae, Coxiellaceae,Cryomorphaceae, Cyclobacteriaceae, Cytophagaceae, Flammeovirgaceae,Flavobacteriaceae, Gammaproteobacteria_family_incertae_sedis,Gemmatimonadaceae, Geobacteraceae, Hydrogenophilaceae,Hyphomicrobiaceae, Ignavibacteriaceae, Lachnospiraceae,Methylobacteriaceae, Methylococcaceae, Micrococcaceae, Nannocystaceae,Nitrosomonadaceae, Nitrospiraceae, Nocardioidaceae, Nocardiopsaceae,OD1_family_incertae_sedis, OP11_family_incertae_sedis, Opitutaceae,Paenibacillaceae_1, Paenibacillaceae_2, Parachlamydiaceae,Pasteuriaceae, Peptostreptococcaceae, Planctomycetaceae, Polyangiaceae,Porphyromonadaceae, Pseudomonadaceae, Pseudonocardiaceae,Puniceicoccaceae, Rhizobiaceae, Rhodobiaceae, Rhodospirillaceae,Rubrobacteraceae, Ruminococcaceae, Saprospiraceae, Simkaniaceae,Sinobacteraceae, Spartobacteria_family_incertae_sedis,Sphaerobacteraceae, Sphingobacteriaceae, Streptococcaceae,Streptosporangiaceae, Subdivision3_family_incertae_sedis,Syntrophorhabdaceae, Thermoactinomycetaceae_1, Thermoactinomycetaceae_2,Thermomonosporaceae, TM7_family_incertae_sedis, Trueperaceae,Verrucomicrobiaceae, Xanthobacteraceae, and Xanthomonadaceae; and genera3_genus_incertae_sedis, Aciditerrimonas, Actinomadura, Actinospica,Adhaeribacter, Aequorivita, Algoriphagus, Alicyclobacillus,Alkanibacter, Alterococcus, Ammoniphilus, Aquicella, Arenibacter,Armatimonadetes_gp2, Armatimonadetes_gp5,Armatimonas_Armatimonadetes_gp1, Arthrobacter, Asticcacaulis,Aureispira, Bacteriovorax, Bdellovibrio, Bellilinea, Blastopirellula,Bosea, BRC1_genera_incertae_sedis, Caldilinea, Cellvibrio, Chondromyces,Clostridium_III, Clostridium_sensu_stricto, Clostridium_XI,Clostridium_XIVa, Cohnella, Conexibacter, Crocinitomix, Cytophaga,Devosia, Dolosigranulum, Dongia, Dyadobacter, Emticicia, Enhygromyxa,Faecalibacterium, Flavobacterium, Fluviicola, Geminicoccus, Gemmata,Gemmatimonas, Geobacter, Gillisia, Gp1, Gp10, Gp16, Gp17, Gp2, Gp21,Gp22, Gp3, Gp4, Gp6, Gp7, Haliscomenobacter, Hymenobacter,lgnavibacterium, Labrys, Lactococcus, Lewinella, Longilinea,Luteolibacter, Lutibacter, Magnetospirillum, Marinobacter,Marinoscillum, Methylobacter, Methylobacterium, Methylocaldum,Nannocystis, Neochiamydia, Nitrosomonas, Nitrospira, Nocardioides,Nonomuraea, OD1_genus_incertae_sedis, Ohtaekwangia,OP11_genus_incertae_sedis, Opitutus, Parachlamydia, Parvibaculum,Pasteuria, Pedobacter, Pelagicoccus, Peredibacter, Petrimonas,Planctomyces, Planifilum, Pontibacter, Prosthecobacter,Proteiniclasticum, Pseudoxanthomonas, Reichenbachiella, Rhizobium,Rhizomicrobium, Rhodanobacter, Rhodomicrobium, Rhodopirellula,Roseomonas, Rubrobacter, Schlesneria, Simkania, Singulisphaera,Skermanella, Solimonas, Sorangium, Spartobacteria_genera_incertae_sedis,Sphaerobacter, Sphingobacterium, Spirosoma, Sporacetigenium, Stella,Steroidobacter, Symbiobacterium, Syntrophorhabdus, Thermoactinomyces,Thermobacillus, Thermobifida, Thermobispora, Thermoflavimicrobium,Thiobacillus, TM7_genus_incertae_sedis, Truepera, Turnebacillus,Verrucomicrobium, Wandonia, Winogradskyella, and Zavarzinella, and atsmall frequencies also genera Acinetobacter, Mycobacterium andLactobacillus.

In an embodiment of the first aspect, the immunomodulatory compositionis free of at least one of the microbial taxa mentioned in the previousembodiment.

In yet another embodiment of the first aspect, the exact frequencies andabundances of microbial taxa mentioned above vary within the sameproduct. The reason is that efficacy of the current invention is atleast partially based on the overall effect of the immunomodulatorycomposition, not on the effect of individual strains.

In an embodiment of the first aspect processing comprises increasing atleast one of usability, comfortability and safety, by using at least oneof the following methods: grinding, sieving, pulverizing and mixing withan aqueous solution or lotion. This step provides the immunomodulatorycomposition in a form, which does not contain sharp edges that caninjure a subject or cause infection.

In an embodiment of the first aspect, the total microbial abundance ofthe subject remains stable but the abundance of at least one microbialphylum, order, class, family, subfamily or genus increases immediatelyafter the treatment on at least one tissue of the subject. In thisembodiment, the increase is caused by at least one non-culturable OTU.

In an embodiment the subject is a mammal, such as an animal subject or ahuman subject, preferably a human subject.

In an embodiment of the first aspect the diversity of the materialderived from nature is at least 3.5 at Shannon index or Shannondiversity index.

In an embodiment of the first aspect the diversity of theimmunomodulatory composition is at least 4 at Shannon index or Shannondiversity index.

In an embodiment the microbial diversity of the immunomodulatorycomposition is indicated at Shannon diversity index by subsampling 2000operational taxonomic units from a 0.25 g wet sample at the 97%similarity level of operational taxonomic units.

In an embodiment the microbial richness of the immunomodulatorycomposition is at least 130 OTUs calculated by rarefying 2000operational taxonomic units from a 0.25 g wet sample at 97% similaritylevel.

In an embodiment the abundance of pathogens in the immunomodulatorycomposition is 0 or at the same or lower level as found in everydayliving environment.

In an embodiment the immunomodulatory composition is biologicallyinactivated before use. Preferably at least one parameter describingrichness, diversity or abundance is determined immediately beforeinactivation, such as any or each of parameters i-iii of the firstaspect or parameter c. of an embodiment of the first aspect.

In an embodiment of the first aspect the richness of theimmunomodulatory composition is at least 350 operational taxonomicunits.

In an embodiment of the first aspect the microbial abundance of theimmunomodulatory composition is at least 1 000 000 000 16 S and 18 Scopies g⁻¹ ww.

In an embodiment of the first aspect the bacterial abundance of theimmunomodulatory composition is at least 1 000 000 000 16 S copies g⁻¹ww.

In a preferable embodiment in the immunomodulatory composition theproportion of non-culturable bacterial 16 S DNA is at least 1% of totalbacterial 16 S DNA. In another embodiment, the proportion ofnon-culturable bacterial 16 S DNA is at least 10%. In a preferableembodiment, the proportion of non-culturable bacterial 16 S DNA is atleast 50%. Immunomodulatory compositions complying with said parameterscomprise a fraction of non-culturable bacteria, which is efficient toprovide immunomodulatory effect in a subject.

In an embodiment in the present immunomodulatory composition the meannumber of 16S copies/g (w/w) is at least 1,000,000,000, preferably atleast 1,500,000,000, more preferably at least 3,000,000,000.

In an embodiment the present immunomodulatory composition is sieved andin it the mean number of 16S copies/g (w/w) is at least 4,000,000,000,preferably at least 5,000,000,000, more preferably at least5,500,000,000.

In an embodiment the diversity of the immunomodulatory composition is ata level or near the level of the material derived from nature. In apreferred embodiment bacterial diversity is essentially the same in thematerial derived from nature and in the immunomodulatory composition. Inanother preferred embodiment bacterial diversity, richness and abundanceare higher in the immunomodulatory composition than in the materialderived from nature. The higher values are a result of processing. In anembodiment, sieved or filtered materials have more surface area per g wwand hence more microbes per g ww than raw materials. In anotherembodiment, a thorough mixing of different processed materials withdifferent microbial communities increases diversity, richness andpotentially also abundance of the resulting new immunomodulatorycomposition so that its diversity, richness and potentially alsoabundance are higher than in materials derived from nature.

In an embodiment of the first aspect the composition further comprisesculturable bacteria. In another embodiment the culturable bacteria areadded to the composition. In yet another embodiment the culturablebacteria comprise Lactobacillus or Mycobacterium.

In an embodiment the material derived from nature is composted. Inanother embodiment the material derived from nature comprises compostedleaves, needles, peat, or a combination thereof. In yet anotherembodiment the level of coliform bacteria is controlled duringcomposting.

In an embodiment the material derived from nature is inactivated. Theinactivation process is selected from heat, radiation, filtration,freezing, chemical treatment or a combination thereof. Preferably theinactivation process is selected such that it provides material which isessentially free from living organisms, but which is able to elicit thesame or similar immunomodulatory effect as the material derived fromnature before inactivation. Example 3 provides an exemplary method totest suitability of the inactivation process for the present invention.

In an embodiment the spatial variation of the microbial community of theimmunomodulatory composition is homogeneous and optionally the densityof pathogens and other pests is controlled.

In an embodiment of the first aspect the processing comprises

-   -   a) homogenizing spatial variation in microbial community;    -   b) controlling pathogens levels and other pests using at least        one inactivation method.

In an embodiment option a) comprises using at least one of the followingmethods: grinding, crushing, sieving, filtering, extraction with anaqueous solution.

In an embodiment option b) comprises using at least one of the followingmethods: homogenization, extraction, evaporation or at least oneinactivation method.

Currently, soil materials, such as composted dressings, are manufacturedfor use in gardening and sieved and mixed coarsely. Then, mesh size isoptimal for gardening purposes i.e. 8 mm or larger, typically 25-35 mm.In the current invention, the mesh size is preferably selected forhomogenizing microbial community i.e. it is less than 8 mm, preferably2-5 mm or even smaller. Optionally sieved material is to be mixedthoroughly, which affects microbial diversity, richness and abundanceand homogenizes spatial variation in microbial community. The processmay be used as a means for controlling the density of pathogens andother pests.

An advantage achieved with the embodiment is that a composition having adefined particle size and good storage stability can be obtained. Anadditional advantage is that the preparation is homogenous and itsquality can be controlled in comparison to preparations based onunprocessed raw materials. Such a composition is easier to administer asaccurate doses and it is more comfortable to use than unprocessed rawmaterials. It is also safer to use, as it does not contain sharp-edgedlarge particles that can injure skin or other tissues.

Importantly, the inactivated or homogenous and quality controlledpreparations have pathogen levels that are at or below those observed ineveryday living environment, as evidenced in examples 5-7 for thecomposition according to the invention. This is a difference of theinventive composition in view of unprocessed raw material such asnatural soil, peat, plant and animal materials and existing organic soilproducts; all these raw materials may contain patches of pathogens thatcan infect humans or toxins derived from pathogens. One of the reasonsfor the high levels of pathogens observed in example 7 is patchydistribution of microbial cells as the non-immunomodulatory compositionsare not homogenized and mixed.

As evidenced by Example 3, the lyophilization and the optionalextraction step preserve the immunomodulatory activity of thecomposition, while providing a composition which has better storagestability and easy application in formulations such as lotions, sprays,cosmetic products and other consumer products and baby and toddlerproducts as shown in example 8.

In an embodiment the immunomodulatory composition has a level ofpathogens encountered in everyday living environment, or an abundance of0.

In another embodiment the pathogens is selected from enterovirus,rhinovirus, rotavirus, norovirus, Giardia and Cryptosporidium.

In an embodiment the level of pathogens is not more than 550 or lesspotentially pathogenic bacterial 16 S sequences per 0.25 g ww sample.

In an embodiment of the first aspect the immunomodulatory composition isessentially free from pathogens. In an embodiment the pathogens areselected from E. coli, Salmonella, Klebsiella or a combination thereof.

In an embodiment the level of pathogens is controlled duringmanufacturing process of the composition. In another embodiment thecontrolling is carried out at a level of genus or species of thepathogen.

In an embodiment of the first aspect the material derived from nature issoil or plant material.

In an embodiment the immunomodulatory composition has a microbialdiversity of at least 3 at Shannon diversity index obtained bysubsampling 2000 operational taxonomic units from a 0.25 g wet sample atthe 97% similarity level of operational taxonomic units.

In an embodiment the immunomodulatory composition has a microbialrichness of at least 130 operational taxonomic units (97% similaritylevel) calculated by rarefying to 2000 sequences from a sample of 0.25 gwet weight of the immunomodulatory composition.

In an embodiment the immunomodulatory composition is inactivated and themicrobial richness, diversity or abundance determined immediately beforethe inactivation.

In an embodiment the microbial community comprises viable non-culturablebacteria and/or immunomodulatory components of viable non-culturablebacteria in the form of inactivated or killed bacteria.

In an embodiment of the first aspect the material derived from nature isselected from soil material; plant material; animal material excludingfeces; microbial material from aqueous environment; sludge; compost;insects; algae; or material stream from industrial, agricultural,silvicultural, water purification, water filtration, aquacultural,mining or peat production, such as a byproduct; and moss from peatproduction areas.

In a preferable embodiment the material derived from nature is soilmaterial and/or plant material.

In an embodiment of the first aspect the immunomodulatory compositioncomprises at least one microbial taxon selected from classesAcidobacteria_Gp1, Acidobacteria_Gp10, Acidobacteria_Gp16,Acidobacteria_Gp17, Acidobacteria_Gp2, Acidobacteria_Gp21,Acidobacteria_Gp22, Acidobacteria_Gp3, Acidobacteria_Gp4,Acidobacteria_Gp6, Acidobacteria_Gp7, Actinobacteria,Alphaproteobacteria, Anaerolineae,Armatimonadetes_gp2_class_incertae_sedis,Armatimonadetes_gp5_class_incertae_ sedis, Armatimonadia, Bacilli,Bacteroidetes_incertae_sedis_class_incertae_sedis, Bacteroidia,Betaproteobacteria, BRC1_class incertae sedis, Caldilineae, Chlamydiae,Clostridia, Deinococci, Deltaproteobacteria, Flavobacteria,Gammaproteobacteria, Gemmatimonadetes, Ignavibacteria, Nitrospira,OD1_class_incertae_sedis, OP11_class_incertae_sedis, Opitutae,Planctomycetacia, Spartobacteria, Sphingobacteria, Subdivision3,Thermomicrobia, TM7_class_incertae_sedis, and Verrucomicrobiae; ordersAcidimicrobiales, Acidobacteria_Gp1_order_incertae_sedis,Acidobacteria_Gp10_order_incertae_sedis,Acidobacteria_Gp16_order_incertae_sedis,Acidobacteria_Gp17_order_incertae_sedis,Acidobacteria_Gp2_order_incertae_sedis,Acidobacteria_Gp21_order_incertae_sedis,Acidobacteria_Gp22_order_incertae_sedis,Acidobacteria_Gp3_order_incertae_sedis,Acidobacteria_Gp4_order_incertae_sedis,Acidobacteria_Gp6_order_incertae_sedis,Acidobacteria_Gp7_order_incertae_sedis, Actinomycetales,Alphaproteobacteria_order_incertae_sedis, Alteromonadales,Anaerolineales, Armatimonadales,Armatimonadetes_gp2_order_incetae_sedis,Armatimonadetes_gp5_order_incetae_sedis, Bacillales, Bacteroidales,Bacteroidetes_incertae_sedis_order_incertae_sedis, Bdellovibrionales,BRC1_order_incertae_sedis, Caldilineales, Caulobacterales, Chlamydiales,Clostridiales, Deinococcales, Deltaproteobacteria_order_incertae_sedis,Desulfuromonadales, Flavobacteriales,Gammaproteobacteria_order_incertae_sedis, Gemmatimonadales,Hydrogenophilales, Ignavibacteriales, Lactobacillales, Legionellales,Methylococcales, Myxococcales, Nitrosomonadales, Nitrospirales,OD1_order_incertae_sedis, OP11_order_incertae_sedis, Opitutales,Planctomycetales, Pseudomonadales, Puniceicoccales, Rhizobiales,Rhodospirillales, Rubrobacterales, Solirubrobacterales,Spartobacteria_order_incertae_sedis, Sphaerobacterales,Sphingobacteriales, Subdivision3_order_incertae_sedis,TM7_order_incertae_sedis, Verrucomicrobiales, and Xanthomonadales;families Acetobacteraceae, Acidimicrobineae_incertae_sedis,Acidobacteria_Gp1_family_incertae_sedis,Acidobacteria_Gp10_family_incertae_sedis,Acidobacteria_Gp16_family_incertae_sedis,Acidobacteria_Gp17_family_incertae_sedis,Acidobacteria_Gp2_family_incertae_sedis,Acidobacteria_Gp21_family_incertae_sedis,Acidobacteria_Gp22_family_incertae_sedis,Acidobacteria_Gp3_family_incertae_sedis,Acidobacteria_Gp4_family_incertae_sedis,Acidobacteria_Gp6_family_incertae_sedis,Acidobacteria_Gp7_family_incertae_sedis, Actinospicaceae,Alicyclobacillaceae, Alphaproteobacteria_family_incertae_sedis,Alteromonadaceae, Anaerolineaceae, Armatimonadaceae,Armatimonadetes_gp2_family_incetae_sedis,Armatimonadetes_gp5_family_incetae_sedis, Bacteriovoracaceae,Bacteroidetes_incertae_sedis_family_incertae_sedis, Bdellovibrionaceae,Bradyrhizobiaceae, BRC1_family_incertae_sedis, Caldilineaceae,Carnobacteriaceae, Caulobacteraceae, Clostridiaceae_1 ,Clostridiales_Incertae_Sedis_XVIII, Conexibacteraceae, Coxiellaceae,Cryomorphaceae, Cyclobacteriaceae, Cytophagaceae, Flammeovirgaceae,Flavobacteriaceae, Gammaproteobacteria_family_incertae_sedis,Gemmatimonadaceae, Geobacteraceae, Hydrogenophilaceae,Hyphomicrobiaceae, Ignavibacteriaceae, Lachnospiraceae,Methylobacteriaceae, Methylococcaceae, Micrococcaceae, Nannocystaceae,Nitrosomonadaceae, Nitrospiraceae, Nocardioidaceae, Nocardiopsaceae,OD1_family_incertae_sedis, OP11_family_incertae_sedis, Opitutaceae,Paenibacillaceae_1, Paenibacillaceae_2, Parachlamydiaceae,Pasteuriaceae, Peptostreptococcaceae, Planctomycetaceae, Polyangiaceae,Porphyromonadaceae, Pseudomonadaceae, Pseudonocardiaceae,Puniceicoccaceae, Rhizobiaceae, Rhodobiaceae, Rhodospirillaceae,Rubrobacteraceae, Ruminococcaceae, Saprospiraceae, Simkaniaceae,Sinobacteraceae, Spartobacteria_family_incertae_sedis,Sphaerobacteraceae, Sphingobacteriaceae, Streptococcaceae,Streptosporangiaceae, Subdivision3_family_incertae_sedis,Syntrophorhabdaceae, Thermoactinomycetaceae_1, Thermoactinomycetaceae_2,Thermomonosporaceae, TM7_family_incertae_sedis, Trueperaceae,Verrucomicrobiaceae, Xanthobacteraceae, and Xanthomonadaceae; and genera3_genus_incertae_sedis, Aciditerrimonas, Actinomadura, Actinospica,Adhaeribacter, Aequorivita, Algoriphagus, Alicyclobacillus,Alkanibacter, Alterococcus, Ammoniphilus, Aquicella, Arenibacter,Armatimonadetes_gp2, Armatimonadetes_gp5, ArmatimonasArmatimonadetes_gp1, Arthrobacter, Asticcacaulis, Aureispira,Bacteriovorax, Bdellovibrio, Bellilinea, Blastopirellula, Bosea,BRC1_genera_incertae sedis, Caldilinea, Cellvibrio, Chondromyces,Clostridium_III, Clostridium_sensu_stricto, Clostridium_XI,Clostridium_XIVa, Cohnella, Conexibacter, Crocinitomix, Cytophaga,Devosia, Dolosigranulum, Dongia, Dyadobacter, Emticicia, Enhygromyxa,Faecalibacterium, Flavobacterium, Fluviicola, Geminicoccus, Gemmata,Gemmatimonas, Geobacter, Gillisia, Gp1, Gp10, Gp16, Gp17, Gp2, Gp21,Gp22, Gp3, Gp4, Gp6, Gp7, Haliscomenobacter, Hymenobacter,Ignavibacterium, Labrys, Lactococcus, Lewinella, Longilinea,Luteoibacter, Lutibacter, Magnetospirillum, Marinobacter, Marinoscillum,Methylobacter, Methylobacterium, Methylocaldum, Nannocystis,Neochlamydia, Nitrosomonas, Nitrospira, Nocardioides, Nonomuraea,OD1_genus_incertae_sedis, Ohtaekwangia, OP11_genus_incertae_sedis,Opitutus, Parachlamydia, Parvibaculum, Pasteuria, Pedobacter,Pelagicoccus, Peredibacter, Petrimonas, Planctomyces, Planifilum,Pontibacter, Prosthecobacter, Proteiniclasticum, Pseudoxanthomonas,Reichenbachiella, Rhizobium, Rhizomicrobium, Rhodanobacter,Rhodomicrobium, Rhodopirellula, Roseomonas, Rubrobacter, Schlesneria,Simkania, Singulisphaera, Skermanella, Solimonas, Sorangium,Spartobacteria_genera_incertae sedis, Sphaerobacter, Sphingobacterium,Spirosoma, Sporacetigenium, Stella, Steroidobacter, Symbiobacterium,Syntrophorhabdus, Thermoactinomyces, Thermobacillus, Thermobifida,Thermobispora, Thermoflavimicrobium, Thiobacillus,TM7_genus_incertae_sedis, Truepera, Tumebacillus, Verrucomicrobium,Wandonia, Winogradskyella and Zavarzinella, Acinetobacter, Mycobacteriumand Lactobacillus.

In an embodiment of the first aspect the immunomodulatory compositionfurther comprises material from at least one eukaryote or virus selectedfrom Fungi, bacteriophage, plant virus, Ecdysozoa, including Nematoda,Arachnida, Acari, Amobae, insects and other multicellular butmicroscopic soil organisms and unicellular eukaryotes such as Amoebozoaand unicellular fungi.

In an embodiment the immunomodulatory composition is in the form of atopical composition, lotion, cream, gel, powder, pill, food ingredient,drink constituent, detergent, conditioner, shampoo, soap, liquid soap,extract, dried extract, freeze-dried extract, spray, steam, water vapor,gas, aerosol or a dry mixture packed inside bags that allow the contactof the immunomodulatory composition with the subject. In anotherembodiment the immunomodulatory composition is in the form of, or packedinside, a container, package or packet, such as a jewel or accessory.

In another embodiment the immunomodulatory composition is in the form ofa chip, granule, microparticle or a combination thereof. In another formthe immunomodulatory composition is in the form of an infusion, steam,water vapor or an aqueous composition comprising active agentsobtainable from the immunomodulatory composition.

In another embodiment the immunomodulatory composition is provided in acarrier. In an embodiment the carrier comprises aqueous vapour, aerosolor liquid.

In an embodiment the immunomodulatory composition is lyophilized and theliquid obtained during lyophilization is recovered.

In an embodiment the immunomodulatory composition is inactivated.Preferably the inactivation process at least partially exposesintracellular components to enhance immune response.

In an embodiment the immunomodulatory composition comprises at least oneadditive. The additive may be selected from a fragrant, preservative,coloring agent, moisturizer, bulking agent or stabilizer.

In an embodiment of the second aspect the use provides a change in themicrobiome of the subject.

In an embodiment of the second aspect the use prevents a negative changein the diversity of the microbiome of the subject.

In an embodiment of the second aspect the exposure comprises inhalation,ingestion, touching or a combination thereof.

In an embodiment the exposure is by ingesting a dose unit comprising theimmunomodulatory composition. In an embodiment the dose unit is a pill,or a capsule.

In an embodiment of the second aspect the exposure is continued for atleast 2 weeks.

In an embodiment the exposure is carried out sequentially at intervalsof not more than 7 days, preferably on daily basis.

In an embodiment of the second aspect the exposure is carried outsequentially at intervals of max 30 days. In another embodiment theexposure is daily. In another embodiment the exposure happens threetimes a day. In yet another embodiment the exposure is continuous andlasts for at least one month.

In an embodiment of the second aspect exposing comprises bringing anarea on the skin of the subject in contact with the immunomodulatorycomposition for at least 1 second. In another embodiment the contact isfor at least 10 s, 30 s, 1 min, 2 min, 3 min, 5 min, 10 min, 30 min, 1h, 2 h, 3 h, 5 h or 12 h. In an embodiment the contact is continuous.The contact time can also be interrupted by a short interval after whichthe contact continues.

In an embodiment the area on the skin of the subject is hand or hands,foot or feet, head, neck, lip or lips, breast or breasts, genital areaor face.

In an embodiment the exposure comprises direct hand exposure with thepresent immunomodulatory composition, or exposure with fabric packetcontaining the present immunomodulatory composition. In an embodimentthe hands are rubbed optionally followed by washing the hands withwater. In an embodiment the packet contains Sphagnum moss as theimmunomodulatory composition.

In an embodiment of the second aspect

-   -   a. the subject has an altered microbial diversity and/or        richness or abundance compared to a reference subject;    -   b. exposing is carried out at least until the microbial        diversity, richness and/or abundance returns towards a level        corresponding to the level of a reference subject or closer to        the level of the reference subject; and    -   c. the reference subject is a subject having a microbial        diversity, richness and/or abundance corresponding to a level        observed for subjects living in a non-urban environment.

Preferably the reference subject has a healthy microbiome, which doesnot expose the reference subject to immunomodulatory disorders.

The use provides the subject with an increased microbial diversity,richness and/or abundance compared to the subject before exposure.

In an embodiment exposing is continued after reaching a predeterminedlevel of microbial diversity, richness and/or abundance to maintain adiverse microbiome.

In an embodiment the internal microbial diversity of the subjectincreases upon exposure to the immunomodulatory composition. In anembodiment the internal microbial diversity is diversity of gutmicrobiome.

In an embodiment the exposure to the immunomodulatory compositionselectively induces immunoregulatory response in the subject. In anembodiment the response is an IL-10 immunoregulatory response, or anIL10-mediated immunomodulatory response. In another embodimentinflammatory response is not increased. In yet another embodiment theresponse is a TGF-β immunoregulatory response, or an TGF-β-mediatedimmunomodulatory response. In another embodiment the immunoregulatoryresponse is both an IL-10 mediated and TGF-β mediated immunomodulatoryresponse.

In an embodiment the subject is a human subject having a disorder ofimmunity or a human subject living in an urban environment whereinmicrobial diversity, richness and/or abundance is lower than in thematerial derived from nature.

In an embodiment of the second aspect the subject is a human subjecthaving a disorder of immunity or a subject having a risk to developimmune related disorder. In an embodiment the risk is due to inadequatemicrobial exposure.

Administering the immunomodulatory composition to subjects in need ofstrengthening their immune system may benefit from the administration.

In an embodiment of the third, fourth or the fifth aspect the drying iscarried out by oven drying.

In an embodiment of the third, fourth or fifth aspect the obtainedproduct is moisturized to provide lotion or liquefied material.

In an embodiment of the third, fourth or fifth aspect the processcomprises an inactivating step.

In an embodiment of the third, fourth or fifth aspect the processcomprises lyophilizing the material obtained in any of steps a-e andtaking it directly to the sterilization step. In another embodiment theraw material is sterilized.

In an embodiment of the third, fourth or the fifth aspect the methodcomprises lyophilizing the material obtained in step b-e and taking itdirectly to the sterilization step.

In an embodiment of the third or the fourth aspect material derived fromnature is extracted. Preferably extraction is carried out using anaqueous solvent, such as water.

In an embodiment of the third, fourth or fifth aspect the methodcomprises lyophilizing the material obtained in step d and taking itdirectly to step f.

In an embodiment of the third, fourth, or fifth aspect material derivedfrom nature comprises plant material and the processing comprisesgrinding.

In another embodiment the material derived from nature comprises plantmaterial and the processing comprises directly extracting materialderived from nature.

In an embodiment of the third, fourth or fifth aspect the methodcomprises lyophilizing the material obtained in step a-d.

In an embodiment of the third aspect the method additionally comprisesat least one of the following steps:

-   -   i. sterilizing the raw material to provide sterilized material;    -   ii. evaporating the raw material to provide evaporated material;    -   iii. Extracting the product obtained in step a., b., c., d., e.,        f., or g. to provide an extract;    -   iv. lyophilizing the extract to provide the lyophilized extract;    -   v. oven-drying the extract to provide oven-dried extract;    -   vi. evaporating the extract to provide gaseous material;    -   vii. condensing the evaporated extract to provide condensed        material;    -   viii. composting the raw material to provide composted material.

In an embodiment of the third, fourth or fifth aspect an inactivationstep is carried out to inactivate pathogens. In a preferred embodimentinactivation is by heat treatment. In another inactivation is carriedout until the level of pathogens is low enough for safe external orinternal use for a human subject.

In an embodiment of the third, fourth or fifth aspect the method furthercomprises grinding the raw material to a particle size of 1000 μm orsmaller. In another embodiment, the particle size is less than 1000 μmin at least 50% of the mass of the immunomodulatory composition, asevidenced in example 10.

In an embodiment of the third, fourth or fifth aspect the method formanufacturing comprises further extracting the lyophilized raw materialwith an aqueous solution or organic solvent, and optionally drying andoptionally resuspending into aqueous solution.

In an embodiment of the fifth aspect the compartment is configured toreceive the composition in at least one unit dose form. In an embodimentthe unit dose is a permeable or semi permeable bag, pouch, patch, packetor a container. In an embodiment the unit dose is made of cotton fabricenclosing the immunomodulatory composition. In another embodiment theunit dose contains immunomodulatory composition in a predeterminedamount, such as a predetermined weight, volume, abundance, richness ordiversity.

In an embodiment of the fifth aspect the compartment comprises a pocketadapted to receive a unit dose of the immunomodulatory composition andadapted to allow exposure of a subject to the immunomodulatorycomposition.

In an embodiment of the fifth aspect the article is selected fromtextile, fabric, a piece of headwear, a hat, sheet, pillow, duvet,blanket, mattress, and baby carrier.

In an embodiment of the fifth aspect the article is selected from

-   -   textile, fabric, a piece of headwear, a hat, sheet, pillow,        duvet, blanket, mattress, baby carrier;    -   tissue paper, diaper, handkerchief, breastfeeding towel,        feminine towel, pad, cushion, underlay;    -   food such as beverage;    -   pharmaceutical or personal care product such as pill, spray,        toothpaste, lipstick, deodorant, mouthwash, talcum;    -   cigarette;    -   toy, building block, security blanket, comfort object, jewel;    -   a piece of cloth, a children's cloth, a baby cloth, or a baby        hat.

In an embodiment of the fifth aspect the article is a piece of cloth. Ina preferred embodiment the cloth is a children's cloth, even morepreferably a baby cloth, such as a baby hat.

In an embodiment of the sixth aspect the apparatus comprises means forproviding aerosol of the immunomodulatory composition.

In an embodiment of the sixth aspect the apparatus is an air refresher,inhalator, nebulizer, or air moisturizer.

In an embodiment of the sixth aspect the apparatus is configured toreceive a cartridge for the immunomodulatory composition. Theimmunomodulatory composition may be provided in liquid form inside thecartridge. The apparatus may further comprise means for evaporating,spraying or nebulizing the immunomodulatory composition.

In an embodiment the immunomodulatory composition is mixed to controlamount of pathogens. By mixing the non-culturable bacteria compete withpathogens and culturable fast growing bacteria, and reduce their amount.

In an embodiment the material derived from nature is of non-humanorigin. In another embodiment the material derived from nature does notcontain raw materials known to contain potential pathogens, such asfeces or dung not treated with antibiotics.

In an embodiment the present immunomodulatory composition is used tospray plants, preferably edible plants. This embodiment is advantageousbecause it can be used to spray for example houseplants with whichpeople are in contact with. It can also be used to spray edible plantsin urban or rural gardens, which enhances their immunomodulatory effectupon use.

EXAMPLES Example 1: Effect on Skin Bacterial Diversity Methods

Sampling and Experiment

Two urban volunteers conducted the experiment. Fourteen differentmaterials derived from nature were modified by sieving differentcomposted, soil and plant based mixtures. The raw materials comprised ofdung, horse dung, chicken dung, deciduous leaf litter, plant debris,horticultural peat, sludge, fine mineral soil such as silt as well ascrushed tree bark mulch. Volunteers rubbed their hands in a testmaterial for 20 seconds, washed their hands without soap in tap waterfor 5 seconds, and dried the hands with hand towels. The procedure wasrepeated until all test materials were tested. Materials were tested inrandom order. No more than two materials were tested in the same day andthere was at least five hours between the tests. Volunteers did not washtheir hands before exposures, in order to validate the changes infrequency, abundance and diversity of skin bacteria.

A skin swab (back of the right hand, 3×3 cm area, 9 wipes) was takentwice, immediately before exposure and immediately after drying handswith a hand towel. A cotton wool stick was first wetted in Tween® 20,used in sampling and cut to a sterile polyethene sample tube.

Sample Preparation to MiSeq Sequencing for Skin Samples

Skin swap samples were stored in deep freezer (<−70° C.) in tubescontaining Tween 20 (MP Biomedicals) (0.1%)+NaCl (0.1 M, J. T.Baker)before DNA extraction. Total DNA was extracted from samples usingPowerSoil® DNA Isolation Kit (MoBio Laboratories, Inc., Carlsbad,Calif., USA) according to the manufacturer's standard protocol. The swapand the liquid (approximately 650 μl Tween+NaCl) were transferred to thePowerBead tube for a homogenization and lysis procedure. DNA was checkedwith agarose gel (1.5%) electrophoresis (120 V, 30 min). Total DNAconcentration was measured with Quant-iT™ PicoGreen® dsDNA reagent kit(Thermo scientific, MA, USA). DNA was analyzed for bacterial (16S)communities using a two-step PCR approach to avoid a 3′-endamplification bias resulting from the sample-specific DNA tags (Berryet. al 2011). The v1-3 regions within the 16S ribosomal RNA (rRNA) genewas amplified by primary PCR (three replicates from each sample) usingpA and PD Illumina primers. Primary PCR was carried out in a reactionmixture (reaction volume 50 μl) consisting of 1 μl each of 10 mMdeoxynucleoside triphosphates (dNTPs; Thermo scientific, MA, USA), 5 μlforward primer pA_Illum_FP (10 μM;ATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTAGAGTTTGATCMTGGCTCAG) and 5 μlreverse primer pD'_Illum_RP (10 μM;GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGTATTACCGCGGCTGCTG), 0.5 μl 2 U/μlPhusion Green Hot Start II High-Fidelity DNA polymerase (Thermoscientific, MA, USA), 10 μl 5× Green HF PCR buffer (F-537), 5 μltemplate DNA and 23.5 μl sterile water. The PCR reaction was run in athermocycler (MJ Research, MA, USA) as follows: initial denaturation at98° C. for 5 min, followed by 30 cycles with denaturation at 94° C. for1 min, annealing for 10 sec at 50° C. and extension for 1 min at 72° C.,and then a final extension at 72° C. for 10 min. The PCR products weredetected with agarose gel (1.5%) electrophoresis (120 V, 1 h). The PCRproducts were purified using Agencourt AMPure XP solution (BeckmanCoulter Ins.).

Illumina adapter overhang nucleotide sequences were added to the 16SrRNA gene-specific sequences in the secondary PCR. The secondary PCR andsequencing was performed at the institute of biotechnology (Universityof Helsinki) using Illumina MiSeq platform. An approximately 500 bpfragment covering the V1-V3 variable regions of the 16S rRNA gene wasamplified and using primers pA (mixture of 5′-AGAGTTTGATCMTGGCTCAG-3′,5′-TAGAGAGTTTGATCMTGGCTCAG-3′, 5′-CTCTAGAGTTTGATCMTGGCTCAG-3′) and pD′(mixture of 5′-GTATTACCGCGGCTGCTG-3′, 5′-CGTATTACCGCGGCTGCTG-3′,5′-TAGTATTACCGCGGCTGCTG-3′). The primers had 5′ overhangs for Illuminasequencing 5′-ATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3′ for pA and5′-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3′ for pD'. The amplification wasdone using a two-step PCR protocol. In the second PCR-step full lengthadapters and Indexes were introduced. The PCR protocol was as describedin Koskinen et.al. (2011). The sequencing was done as paired-end (300bp+300 bp) on a MiSeq Illumina instrument using a v3 reagent kit.

Bacterial Abundance—qPCR Method

The q-PCRs of bacterial 16S rRNA were based on SYBR green detection.PCRs were carried out with the Light Cycler 96 Quantitative real-timePCR machine (MJ Research, MA, USA). The forward primer used was pE5′-AAA CTC AAA GGA ATT GAC GG-3′ (SEQ ID NO:1) and the reverse primer pF5′-ACG AGC TGA CGA CAG CCA TG-3′ (SEQ ID NO: 2) (Oqvist et al. 2008).All samples were run in triplicates in 20 μl reactions containing 10 μl2× PowerUp SYBR Green Master Mix (Thermo scientific, MA, USA), 0.2 μl 20mg/ml BSA, 0.5 μl of each primer (10 μM), and the sample template. Astandard curve was included in every run to allow quantitation of thenumber of bacterial 16S copies present in the original sample. The q-PCRrun was as follows: initial denaturation at 95° C. for 2 min, followedby 40 cycles of denaturation at 95° C. for 10 s, annealing for 20 sec at53° C. and extension for 30 s at 72° C. Melting curve analysis on theamplicon was as follows: 95° C. for 10 s, 65° C. for 60 s, 97° C. for 1s, 37° C. for 30 s with continuous measurement of the fluorescencesignal. DNA of Cupriavidus necator JMP134 (DSM 4058) was used as thestandard. The standard worked also as a positive control while sterilewater was used as a negative control.

Sequence Processing

We analyzed the sequence data using Mothur-program (versions 1.36.1;Schloss et al. 2009). The sequence processing protocol partly followedthe pipeline suggested by Schloss et al. (2011) and (Kozich et al.2013). The paired sequences contained in reverse and forward fastq fileswere aligned into a contig. Sequences were trimmed and screened toremove any mismatches with primer or DNA-tag sequences, ambiguous basesand homopolymers larger than 8 bp long. Sequences were aligned usingMothur version of SILVA bacterial reference sequences (version 102;Pruesse et al. 2007) and the sequences which were not aligned to areference alignment of the correct sequencing region were removed.Unique sequences and their frequency in each sample were identified, andthen, almost identical sequences (>99% similar) were preclustered tominimize sequencing errors (Huse et al. 2010) and screened for chimeras(UCHIME, Edgar et al. 2011) using the abundant sequences as a reference.The chimeric sequences were removed. We calculated a pairwise distancematrix for unique sequences and clustered OTUs at 97% sequencesimilarity using the nearest neighbor algorithms. Sequences wereclassified using the Mothur version of Bayesian classifier (Wang et al.2007) with the RDP training set version 9 (Cole et al 2009). Sequencesclassified to Chloroplast, Mitochondria, unknown, Archaea and Eukaryotawere removed from the analyses. Rare OTUs that were represented with 10or fewer sequences in the whole data were removed. Finally, all thesamples were rarefied to 7388 sequences which was the minimum totalnumber of sequences in a sample.

Statistical Methods

The Wilcoxon signed-rank test was used for comparing the number ofbacterial 16S copies in hands before and immediately after exposure. TheWilcoxon signed-rank test is used for comparing two related samples whenthe population cannot be assumed to be normally distributed. Wilcoxonsigned-rank test was also used for comparing taxonomic richness andShannon diversity index before and after exposure.

The difference between bacterial composition in hands before andimmediately after exposure was studied using permutational multivariateanalysis of variance (PERMANOVA; Anderson 2001). PERMANOVA was run usingR package vegan and a function adonis. This analysis was not run forAcidobacteria because one of the samples did not include anyAcidobacteria. Analysis was based on Bray-Curtis distance and it wasconducted for both relative abundance data as well as forpresence-absence data.

Results

Wilcoxon signed rank test showed that number of bacterial 16S copies wassignificantly larger after the exposure on immunomodulatory compositionscompared to the number of copies before exposure (p-value=0.001709, V=6)(FIG. 1).

Richness (FIG. 2) and Shannon diversity index (FIG. 3) weresignificantly (p<0.05) higher after exposure in all tested taxonomicgroups excluding the difference in richness for phylum Firmicutes.PERMANOVA showed that bacterial composition was significantly differentin hands after exposure compared to the bacterial composition beforeexposure in all tested taxonomic groups (Table 1).

TABLE 1 P-values for the difference between bacterial composition inhands before and after exposure to immunomodulatory compositions basedon permutational multivariate analysis of variance (PERMANOVA). Analysiswas based on Bray-Curtis distance for relative abundances andpresence-absence data. Analysis was not run for Acidobacteria becauseone of the samples did not include any Acidobacteria. Presence-Abundance absence data data OTUs all 0.001 0.001 TAXA Genus 0.001 0.001TAXA Family 0.001 0.001 TAXA Order 0.001 0.001 TAXA Class 0.001 0.001TAXA Phylum 0.002 0.001 OTUs within Phylum Actinobacteria 0.001 0.001OTUs within Phylum Bacteroidetes 0.002 0.002 OTUs within PhylumFirmicutes 0.007 0.001 OTUs within Phylum Proteobacteria 0.001 0.001OTUs within Phylum unclassified 0.001 0.001 OTUs within ClassAlphaproteobacteria 0.001 0.001 OTUs within Class Betaproteobacteria0.001 0.002 OTUs within Class Gammaproteobacteria 0.003 0.001

As the results show, bacterial abundance, richness and diversity on skincould be increased after exposure to the immunomodulatory compositionaccording to the invention. The effect was found at different taxonomiclevels in the whole bacterial community, and within major phyla andclasses. As the results further show (Table 1), the immunomodulatorycompositions also changed bacterial community composition in hands. Thetechnical effect is that the current invention is suitable forincreasing bacterial abundance, richness and diversity of urbansubjects.

Example 2—Effects of Immunomodulatory Composition on Fecal DiversityMethods

Experimental Groups, Exposure and Sampling

Fourteen urban volunteers (healthy adults, age 27-63 years) participatedin the experiment, which followed a case-control design. The pairs werematched for sex, age (difference max 11 years), pet ownership (no pet ordog), and dwelling type (apartment building, row house, detached house).One person of the pair was randomly picked to be part of the case groupwhile the other became part of the control group.

The case group conducted two weeks' exposure, i.e. they rubbed theirhands in a soil and plant based immunomodulatory composition three timesevery day: before breakfast, before dinner/evening snack, and beforegoing to bed. Each exposure lasted for 10-20 seconds after which thestudy subjects were instructed to wash their hands with tap water butwithout soap for five seconds and pad their hands dry with a towel.

The soil and plant based immunomodulatory composition was manufacturedby sieving and combining various commercially available, composted soilmaterials. The general ingredients for these soil-types were variouscompositions of industrial composts (raw materials cattle dung, horsedung, chicken dung, deciduous leaf litter, plant debris, horticulturalpeat, sludge, fine mineral soil such as silt as well as crushed treebark mulch). The immunomodulatory composition consisted of four majoringredients (three types of composted soil materials and dried Sphagnummoss) and eight minor ingredients (peat and different composted soilmaterials). Before mixing, the major and minor components were sievedwith ∅ 5 mm and ∅ 2 mm sieves, respectively, except for the moss thatwas dried and crushed and mixed. The combined ratio for the testmaterial was 4:1:0.5 for each major composted materials, moss and eachminor materials, respectively.

Stool samples were collected from the study subjects just before thestart of the exposure (day 0), immediately after the exposure period(day 14), and three weeks after the exposure had ended (day 35). Allstudy subjects were given a Pneumococcus vaccination immediately afterthe two weeks' exposure period.

Sample Preparation to MiSeq Sequencing for Stool Samples

The total DNA was extracted from 30-60 milligrams of frozen unprocessedstool sample using the PowerSoil DNA isolation kit (MoBio, Carlsbad,Calif.) according to manufacturer's protocol. One water sample was addedinto each extraction batch.

The microbiome was assessed using the variable region 4 of the 16Sbacterial ribosomal gene. Amplification was done using 17 μl of AccuPrime Pfx SuperMix (Invitrogen, Waltham, Mass.), 1 μl of template DNAand μl of paired set of index primers (Schloss et al. 2011).Amplification was initiated with denaturation at 95° C. for 5 minutes,followed by 30 cycles of denaturation, 95° C., 15 seconds, primerannealing, 55° C., 30 seconds and extension, 68° C., 1 minute. Productswere checked by 1% agarose gel electrophoresis (100V for 30 minutes).PCR products were diluted in water and purified with Ampure magneticbeads (Beckman Coulter, Fullerton, Calif.). The PCR products werequantified with KAPA Library quantification kit (Kapa Biosystems,Wilmington, Mass.) and further were equalized according to the results.Mock community, which is a mix of bacterial DNA of a known content, wasused as positive control for next generation sequencing (NGS).

Sequencing was performed on a MiSeq machine (Illumina, San Diego,Calif.) with a 2×250 bp version 2 sequencing kit according tomanufacturer's protocol.

Sequence Processing

Stool NGS data was processed and analyzed using mothur (version 1.36.1;Schloss et al. 2009), custom python scripts, and QIIME (Caporaso et al.,2010). The sequence processing protocol partly followed the pipelinesuggested by Schloss et al. (2011) and (Cinek et al., 2016). The pairedsequences contained in reverse and forward fastq files were aligned intoa contig. Sequences were trimmed and screened to remove any mismatcheswith primer or DNA-tag sequences, ambiguous bases and homopolymerslarger than 8 bp long. Sequences were aligned using Mothur version ofSILVA bacterial reference sequences (version 102; Pruesse et al. 2007)and the sequences which were not aligned to a reference alignment of thecorrect sequencing region were removed. Unique sequences and theirfrequency in each sample were identified and screened for chimeras(usearch academic version, http://www.drive5.com/usearch) using theabundant sequences as a reference. The chimeric sequences were removed.We calculated a pairwise distance matrix for unique sequences andclustered OTUs at 97% sequence similarity using the nearest neighboralgorithms. Sequences were classified using the Mothur version ofBayesian classifier (Wang et al. 2007) with the RDP training set version9 (Cole et al 2009). Sequences classified to Chloroplast, Mitochondria,unknown, Archaea and Eukaryota were removed from the analyses. Greengenes (DeSantis et al., 2006) core imputed reference was furtherintegrated for building phylogenetic tree required for furtherdownstream statistical analysis.

Statistical Methods

The R language platform was used for statistical testing and plotting.Key packages include the microbial package Phyloseq V16.2 (McMurdie &Holmes, 2013) and fold change estimation Deseq2 (Love, Huber, & Anders,2014). The purpose of statistical analyses was to find out if theimmunomodulatory composition changes richness and ecosystem diversity infecal samples. Differences in fecal bacterial richness between cases andcontrols were analyzed using Fisher diversity index i.e. Fisher's alpha(Magurran 2004). Ecosystem diversity was tested using Bray-Curtisdissimilarity data (Legendre and Legendre 1998). Changes and rates ofchange between the start (day 0) and the end (day 14) of the exposurewere especially important, as the composition change should be directlycomparable to the exposure effects on gut microbiome.

Subsampling was accomplished using phyloseq function rarefy_even_depthwith sample sum of 2000 reads to filter rare OTUs and at the same timeto preserve maximal bacterial diversity representation. All sampleswithin case and controls passed this threshold. The difference betweenbacterial composition in stool before and after exposure was calculatedusing rate of change in sample diversity between days 0 and 14. Thesecond measure was ecosystem diversity (Bray-Curtis) dissimilarityscores between days 0 and 14.

Rate of change of Fisher diversity index and ecosystem diversitymeasured as Bray-Curtis dissimilarity were divided to two equally largegroups for Fisher's exact test (Fisher 1954).

Medians were used as cut points so that values below median formed onegroup (n=7) and values above median another group (n=7). Thereaftercases and controls were compared.

Results and Interpretation

We focus on sample species richness and ecosystem diversitydissimilarities results related to change over time points inconcordance with experiment design.

Fisher diversity index values of sample species richness varied between0.027 and 0.084. There were no differences between cases and controls inthe actual values, but the rate of change from day 0 to day 14 wasdifferent (Table 2). Six out of seven cases belonged to the above mediangroup and therefore the p-value in Fisher's exact test was 0.029. Table2 thus shows the increased sample diversity (6 out of 7 cases) from day0 to day 14, using Fisher diversity index.

Values of Bray-Curtis dissimilarity index of ecosystem diversity variedbetween 0.21 and 0.46. The values were higher in the case than thecontrol group. Six out of seven cases belonged to the above median groupand therefore the p-value in Fisher's exact test was 0.029. Table 2 thusshows that the case group exhibited higher amount of species change(6/7). To illustrate the difference between cases and controls, FIG. 4shows ecosystem diversity as measured with Bray-Curtis distance withNMDS ordinates. The graph nodes are labeled with time and case/controlvariables.

As the results confirm, volunteer subjects exposed to theimmunomodulatory composition, from day 0 to day 14, had higher rates ofchange in species richness and higher amount of species change in stoolsamples compared to non-exposed volunteers. This is an impactfulrevelation as gut microbiota has been linked to numerous immune diseasesincluding inflamed bowel systems, type 1 diabetes, and Crohn's disease.A technical effect is that the external use of the immunomodulatorycomposition as described herein can lead to an internal increase inmicrobial diversity.

TABLE 2 Sample richness changes as measured by rate of change in Fisherdiversity index and ecosystem diversity as measured by Bray-Curtisdissimilarity between days 0 and 14. Rate of Rate of Ecosystem Ecosystemchange, change, diversity, diversity, Pairs Case Control Case Control 10.032 0.027 0.399 0.369 2 0.130 0.013 0.397 0.290 3 0.041 −0.073 0.2170.267 4 0.078 −0.073 0.322 0.229 5 −0.034 0.024 0.301 0.288 6 0.0320.089 0.384 0.253 7 0.081 −0.039 0.462 0.257 P = 0.029 in Fisher’s exacttest for both variables.

Example 3—Peripheral Blood Mononuclear Cell Stimulation with Extractedand Freeze Fried Immunomodulatory Composition Methods

Approximately 1 liter of sieved immunomodulatory composition (details inExample 2) was taken in a clean plastic container. Ultra-pure Milli-Qwater was poured slowly into the container and mixed thoroughly untilthe soil was saturated (i.e. water started dripping when the wet soilwas held in hand). Approximately 800 ml water was needed for saturation.The soil water mixture was kept inside a laminar hood at roomtemperature for 4 hours covered with a lid but holes on the side wallsof the container allowed adequate air circulation. The soil-watermixture was then hand-squeezed using sterilized laboratory gloves overan ethanol-cleaned 250 μm sieve placed above another sterile plasticcontainer. The extract was collected in separate 50 ml Falcon tubes. Allthe samples were frozen at −20° C. prior to freeze-drying. Approximately48 hours was needed for the freeze drying process to complete.

The extracted and freeze fried immunomodulatory composition wasresuspended in the original volume of MilliQ water. The suspendedextract was clarified by low speed centrifugation (5 min 50 g) andfiltered with 35 μm filter. The supernatant was directly used in thestimulation tests. Alternatively it was heat inactivated by 5 minutestreatment at 120° C. or was further filtered twice using first 1 μm andsubsequently 0.45 μm filters. Both heat inactivated and filteredextracts were also used in stimulation tests. In addition, a mixture ofanti-CD3 and anti-CD28 antibodies was used as a positive control that isknown to stimulate immune responses in white blood cells.

Blood was drawn into CPT tubes (Sodium Citrate Vacutainer® CPT™Mononuclear Cell Preparation Tube, BD) and centrifuged according to thetube manufacturers' instructions. Peripheral blood mononuclear cells(PBMCs) were isolated from the tubes, washed twice with RPMI 1640 medium(Life Technologies, Carlsbad, Calif., USA) and placed at 2× 106 cells/mLin RPMI 1640 supplemented with 10% human AB serum (inactivated,Sigma-Aldrich, St. Louis, Mo., United States), 1%penicillin-streptomycin (PAA Laboratories, Pasching, Austria), and 1%L-glutamine (Life Technologies, Carlsbad, Calif., USA). Dilutions of theimmunomodulatory composition and stimulant suspensions were prepared insupplemented RPMI medium, added to the PBMC containing wells andincubated for 24 or 48 hours at 37° C. and 5% CO2. Final concentrationsof control stimulants in the wells were 5 and 0.5 μg/mL for CD3 andCD28, respectively. The cell culture supernatants were collected andIFN-gamma and IL-10 levels were measured in supernatants with ELISA(eBioscience, San Diego, Calif., USA) according to manufacturers'instructions.

Results and Interpretation

FIG. 5A. panel A shows the IL-10 expression in PBMCs induced by theextracted, freeze-dried and resuspended immunomodulatory compositiondetermined at 24 and 48 hours post treatment time points. The positivecontrol consisted of anti-CD3/28 antibodies. Both non-treated and heattreated immunomodulatory compositions gave strong interleukin-10 (IL-10)induction down to dilution 1/100 and to lesser at 1/1000 dilution atboth time points. In contrast, the induction of the filtered compositionwas lower and only detectable in dilution 1/100.

FIG. 5B. panel B shows that the extracted, freeze-dried and resuspendedimmunomodulatory composition induce a lower interferon-gamma (IFN-g)expression compared to positive anti-CD3/28 control. The induction wasdetected only at dilution 1/10 for non-treated soil mixture at 48 h timepoint and weakly for heat treated immunomodulatory composition atdilution 1/10 at both studied time points. All other treatments anddilutions were not able to generate any induction compared to negativecontrol.

The results show that the extracted, freeze-dried and resuspendedimmunomodulatory composition is able to induce a robust immunoregulatoryIL-10 response in white blood cells, whereas the proinflammatoryIFN-gamma response is much weaker. It is possible to find a suitabledosage of the immunomodulatory composition to induce immunoregulatoryresponse without inducing inflammation. This also shows that both intact(living) and heat inactivated immunomodulatory composition are able toinduce a beneficial immunoregulatory response in white blood cells. Incontrast, the ability of filtered composition which lacks bacteria toinduce IL-10 was clearly decreased. The technical effect is that theimmunomodulatory composition according to the current invention caninduce immunoregulatory response without inducing inflammation, and thisproperty depends on the presence of bacteria in this composition. Hence,the immunomodulatory compositions according to the current invention areable to induce a strong immunoregulatory response, which is beneficialto prevent or treat immune mediated disorders and to activate immunesystem in a beneficial way.

Example 4—Comparison of Richness, Diversity and Abundance in UrbanMineral Soils, Materials Derived from Nature and ImmunomodulatoryCompositions Methods

Materials Compared

Immunomodulatory compositions comprising non-culturable microbialcommunity, their raw materials, mineral soil materials and naturalorganic soils were compared to find out potential differences inmicrobial richness, diversity and abundance.

Organic soil materials derived from nature were manufactured as inexample 1. They were divided to two separate groups, those thatcomprised only non-coniferous composted materials derived from nature(called jointly as Organic materials—non-coniferous), and those thatcontained also peat or coniferous plant parts (called jointly as Organicmaterials—coniferous). Sieved immunomodulatory composition wasmanufactured as in example 2. Sieved immunomodulatory compositionconsisted of two separate mixing batches that were not manufacturedsimultaneously. Freeze dried immunomodulatory composition wasmanufactured as in example 3. Organic raw materials derived from natureconsisted of Sphagnum mosses of different geographic origin (3 samples),peat of different geographic origin (2 samples) and two types of woodmulch. Pieces of natural forest soil (1 m² each) were taken from twosources of different geographic origin. They were cut from forest andtransferred immediately to laboratory where sampling was made.Commercially available mineral soil materials were received from RudusOy in May 2016. The materials consisted of playsand 0/2 mm, playgroundgravel ⅛ mm, stone dust 0/3 mm, coarse gravel ⅖ mm, and coarse sand 0/8mm.

Sampling of the Sieved Immunomodulatory Composition

Preparation of the sieved immunomodulatory composition is described inexample 2. Altogether eight samples were taken. Four samples were takenfrom each batch of the immunomodulatory composition. As sievedimmunomodulatory composition was divided into 5 I buckets beforesampling, each sample was taken from a separate bucket. After sampling,the buckets were distributed to study subjects in the case group ofexample 2.

Sampling of the Freeze Dried Immunomodulatory Composition

Freeze dried extract was prepared following the same method described inExample 3, i.e. the sieved immunomodulatory composition described abovewas used. Three samples of the freeze dried extract were used forextraction and sequencing. Additional 3 samples were also used in qPCR.

Sampling of Materials Derived from Nature

The materials derived from nature were mixed thoroughly before sampling.Samples were taken from 5 separate spots. Distance between spots was atleast 3 cm. Sample size was 2 g. They consisted of four types of organicmaterials. Seven samples belonged to the group Organicsoil—non-coniferous, and seven to the group Organic soil—coniferous.Seven samples were organic raw materials derived from silvicultural andpeat production activities (Organic raw material). Natural forest soilswere sampled as follows: sample size 2 g, 5 subsamples per soil, thedistance between sampling points was at least 10 cm, subsamples werepooled and mixed thoroughly before molecular analyses.

Sampling of Mineral Soils

Mineral soil types were sampled separately. Sampling was done with asterile polyethene spoon and the sample was freezed in a cleanpolyethene freezer bag. All the materials are used particularly inplanning of urban spaces e.g. outdoor areas of daycare centers.

Sample Preparation to MiSeq Sequencing

Sample preparation to MiSeq sequencing was done according to Veach etal. (2015). Samples were stored in deep freezer (<−70° C.) before DNAextraction. For each sample, approximately 0.25 g soil was used for DNAextraction. For organic soils, three sample replicates were extractedand pooled before sequencing. For freeze dried extract and mineralsoils, no replicates were used. Total DNA was extracted from samplesusing PowerSoil® DNA Isolation Kit (MoBio Laboratories, Inc., Carlsbad,Calif., USA) according to the manufacturer's standard protocol. Threeorganic soil samples were extracted using PowerMax® DNA Isolation Kit(one sample from each of the following groups: Organic soil—coniferous,Organic raw material and Natural forest). DNA was checked with agarosegel (1.5%) electrophoresis (120 V, 30 min). Total DNA concentration wasmeasured with Quant-iT™ PicoGreen® dsDNA reagent kit (Thermo scientific,MA, USA). The DNA concentration was adjusted to 0.35-0.4 ng/μl to eachsample. DNA was analyzed for bacterial (16S) communities using atwo-step PCR approach to avoid a 3′-end amplification bias resultingfrom the sample-specific DNA tags (Berry et al. 2011). The V4 regionwithin the 16S ribosomal RNA (rRNA) gene was amplified by primary PCR astriplicates using 505F and 806R primers (Caporaso et al. 2012). PrimaryPCR was carried out in a reaction mixture (reaction volume 50 μl)consisting of 1 μl each of 10 mM deoxynucleoside triphosphates (dNTPs;Thermo scientific, MA, USA) 5 μl forward primer 505F (10 μM;5′-GTGCCAGCMGCCGCGGTAA-3′) and 5 μl reverse primer 806R (10 μM; 5′-GGACTACHVGGGTWTCTAAT-3′), 0.5 μl 2 U/μl Phusion Green Hot Start IIHigh-Fidelity DNA polymerase (Thermo scientific, MA, USA), 10 μl 5×Green HF PCR buffer (F-537), 5 μl template DNA and 23.5 μl sterilewater. The PCR reaction was run in a thermocycler (MJ Research, MA, USA)as follows: initial denaturation at 98° C. for 5 min, followed by 25cycles with denaturation at 94° C. for 1 min, annealing for 10 sec at50° C. and extension for 1 min at 72° C., and then a final extension at72° C. for 10 min. A positive control (Cupriavidus necator JMP134, DSM4058) was included in PRC runs to ensure that the PCR worked, and anegative control (sterile water) was run to detect any possiblecontamination. DNA was detected with agarose gel (1.5%) electrophoresis(120 V, 1 h). The PCR products were purified using Agencourt AMPure XPsolution (Beckman Coulter Ins.) to reduce carryover of primary PCRprimers. Triplicates of the cleaned amplicons were pooled and diluted1:5.

Cleaned and diluted primary PCR products were targeted in the secondaryPCR (TagPCR). Reaction mixture to the TagPCR was equal as above exceptreverse primer included a 12 bp unique Multiplexing Identifier tag(MID-806R). Amplification program was the same as above except therewere only seven cycles for soil products and ten cycles for othersamples. TagPCR products were detected on agarose gel (1.5%)electrophoresis (120 V, 1 h), purified with Agencourt AMPure, pooled andthe DNA concentration was measured with PicoGreen. The sequencing wasperformed at the Kansas State University using Illumina MiSeq platform.The sequencing was performed using Illumina MiSeq platform with a 2×300bp version 3 kit sequencing kit according to manufacturer's protocol.The GeneRead DNA Library I Core Kit (Qiagen, catalog #180432) was usedto ligate Illumina's TruSeq adapters to amplicons.

qPCR Method

Quantitative PCR was conducted following same method described inExample 1.

Sequence Processing

We analyzed the sequence data using mothur-program (version 1.35.1 fororganic soils and v.1.38.1 for other samples; Schloss et al. 2009). Thesequence processing protocol partly followed the pipeline suggested bySchloss et al. (2011) and (Kozich et al. 2013). The paired sequencescontained in reverse and forward fastq files were aligned into a contig.The resulted library was trimmed and screened to remove any mismatcheswith primer or DNA-tag sequences, ambiguous bases and homopolymerslarger than 8 bp long. Sequences were aligned using Mothur version ofSILVA bacterial reference sequences (version 102; Pruesse et al. 2007)and the sequences which were not aligned to a reference alignment of thecorrect sequencing region were removed. The samples having more than 20000 sequences were rarefied to 20 000 sequences. At this point ofsequence processing, the samples having less than 20 000 sequences wereretained without rarefying. Unique sequences and their frequency in eachsample were identified, and then, almost identical sequences (>99%similar) were preclustered to minimize sequencing errors (Huse et al.2010) and screened for chimeras (UCHIME, Edgar et al. 2011) using theabundant sequences as a reference. The chimeric sequences were removed.The sequences were classified using Mothur version of Bayesianclassifier (Wang et al. 2007) with the RDP training set version 9 (Coleet al 2009). Sequences that were classified as Mitochondria,Chloroplast, Archaea, Eukaryota or unknown were removed. Operationaltaxonomic units (OTUs) were assigned at 97% identity. Rare OTUs thatwere represented with 10 or fewer sequences in the whole data wereremoved. Some contamination was evident based on controls and thus thesamples were adjusted based on the number sequences in each OTU thatwere found in controls. This was done by taking into account the initialrarefaction to 20 000 sequences of some samples. First, the sample wiseproportions for each OTU were calculated. Second, the expected number ofsequences for each OTU without the initial rarefaction was calculatedusing the proportions and the total number of sequences in each sampleprior the initial subsampling. Third, for each OTU the number ofsequences detected in control was subtracted from the samples andnegative values were changed to zeros. Fourth, the proportion ofsequences removed from each sample was detected and this proportion ofsequences was removed from the data. Finally, all the samples wererarefied to 2000 sequences which is a compromise to have adequate numberof sequences but not to lose too many samples. Three mineral soilsamples had fewer number of sequences and were thus removed in thisstage.

Results

TABLE 3 Number of OTUs i.e. bacterial richness was the highest inimmunomodulatory compositions and the lowest in mineral soils. Materialsderived from nature and natural forest soil had higher values thanmineral soil, but most of them were lower than in immunomodulatorycompositions. Number of OTUs N Min Max Mean StdDev Freeze driedimmunomodulatory 3 436 485 462 25 composition Sieved immunomodulatory 8303 413 361 38 composition Organic soil - coniferous 7 194 377 297 66Organic soil - non coniferous 7 234 431 295 75 Organic raw material 7173 270 226 40 Moss 3 173 270 229 50 Peat 2 216 239 228 16 Wood mulch 2174 268 221 66 Natural forest soil 2 201 248 225 33 Mineral soil -aggregate producer 2 111 115 113 3 N = number of samples.

TABLE 4 Bacterial diversity measured as Shannon diversity index was thehighest in immunomodulatory compositions and the lowest in mineralsoils. Materials derived from nature and natural forest soil had highervalues than mineral soil, but most of them were lower than inimmunomodulatory compositions. Shannon diversity index N Min Max MeanStdDev Freeze dried immunomodulatory 3 5.13 5.32 5.23 0.09 compositionSieved immunomodulatory 8 4.26 4.96 4.66 0.22 composition Organic soil -non coniferous 7 3.81 5.04 4.28 0.46 Organic soil - coniferous 7 3.274.88 4.18 0.58 Natural forest soil 2 3.42 3.86 3.64 0.31 Organic rawmaterial 7 2.51 4.13 3.55 0.57 Moss 3 3.23 4.05 3.73 0.44 Peat 2 3.413.64 3.52 0.16 Wood mulch 2 2.51 4.13 3.32 1.14 Mineral soil - aggregateproducer 2 2.00 2.23 2.11 0.16 N = number of samples.

TABLE 5 Number of 16S copies i.e. bacterial abundance was the highest inimmunomodulatory compositions and some organic soil materials and thelowest in mineral soils. Number of 16S copies/g ww N Min Max Mean StdDevOrganic soil - non coniferous 7 2266053616 11597309791 65001951423477329482 Sieved immunomodulatory 8 2973155588 8992822967 55238955832344838596 composition Natural forest soil 2 4251867322 48599121274555889725 429952605 Freeze dried immunomodulatory 6 22583839754298476160 3462470836 820174162 composition Sequenced samples 32258383975 3817652251 3203707982 830776015 Other samples 3 26972594404298476160 3721233691 889185419 Organic raw material 7 27353318843828910 1874346153 3450616262 Moss 3 834284072 8843828910 36562489474498366725 Peat 2 134146375 140448369 137297372 4456183 Wood mulch 22735331 2735331 2735331 — Organic soil - coniferous 7 4720000002356536589 1571529885 646043086 Mineral soil - aggregate producer 522064 699967 179643 292351 Sieved immunomodulatory 1 16436 16436 16436 —composition - neg. control Freeze dried neg. Control 1 24640 24640 24640— Mineral soil neg. Control 1 9706 9706 9706 — N = number of samples.

As evidenced in Tables 3-5, microbial richness i.e. number of OTUs,diversity i.e. Shannon index and abundance i.e. number of 16S copies arehigher in immunomodulatory compositions than commercially availablemineral soil materials designed for urban planning. The technical effectis that immunomodulatory compositions—if used as defined in theembodiments of the current invention—increase microbial richness,diversity and abundance of a subject, preferably a human subject inurban environment. Hence, immunomodulatory composition according to thecurrent invention had high microbial richness, diversity and abundance,which is beneficial to prevent or treat immune mediated disorders and toactivate immune system in a beneficial way. Importantly, mean diversity,richness and abundance in mineral soils from aggregate producer arelower than in claims 1-2. As these and similar materials are commonlyused in the living environment of urban subjects, the technical effectof the immunomodulatory compositions is that they change microbiota ofurban subjects.

Example 5—Testing Viral and Protozoan Pathogens in Soil Samples Methods

Immunomodulatory compositions presented in examples 1 and 2 were testedfor viral and protozoan pathogens by Q-PCR. Samples were extracted byPowerSoil DNA Isolation Kit or PowerSoil Total RNA Isolation Kit.Samples were tested for enterovirus, rhinovirus, rotavirus, norovirus,Giardia and Cryptosporidium by Q-PCR as described in (Krogvold et al.,2015) Cut-off limit for positive sample was less than 42 Ct-value.Validity of Q-PCR tests was monitored by spiking known amount of targetRNA in the samples. Spiked samples were analyzed by Q-PCR and resultswere compared to positive control.

Results and Interpretation

All samples were negative for tested viruses and parasites. The resultindicates that used manufacture procedures produce material safe for usesince it is free of studied viral and protozoan human pathogens.

Example 6—Pseudomonas Pathogens

The sieved immunomodulatory composition described in example 2 and thefreeze dried immunomodulatory composition described in example 3 weretested for the presence of Pseudomonas aeruginosa with a selectivemedium containing cetrimide (0.3 g/L). Cetrimide agar is commonly andsuccessfully used as a selective medium for the isolation of Pseudomonasaeruginosa from various sources as only this species of Pseudomonas iscapable of producing fluorescein and pyocyanin pigments when grown oncetrimide. In other words, while Pseudomonas aeruginosa forms bluecolonies, other species of Pseudomonas from white colonies. Bacteriathat do not belong to Pseudomonas are inhibited by the presence ofcetrimide on the growth medium.

Test Material Dilutions, Plating and Incubation:

Ten grams of the sieved immunomodulatory composition was weighed into 95ml of milliQ water, and the mixture was shaken vigorously for fewminutes (dilution A=10⁻¹). From this a dilution series of 4 dilutions(B=10⁻²,C=10⁻³,D=10⁻⁴, and E=10⁻⁵) were made by measuring 9 ml of milliQwater into 15 ml Falcon tube and adding 1 ml of previous dilution to thetube thus giving a 100 fold dilution in each step. Each dilution wasvortexed for few seconds and 100 μL was plated on a cetrimide plate(Sigma cat #22470) using a standard sterile technique. Three replicatesof each dilution were plated.

The freeze dried immunomodulatory composition was dissolved in ˜45 ml ofmilliQ water and vortexed lightly into a homogenous suspension. This wascentrifuged for 5 minutes (50×g), and the supernatant was filtered witha 10 μM filter (Whatman cyclopore track etched membrane cat #7060-4715).The filtered immunomodulatory composition was then diluted and plated asdescribed above.

A part of the filtered immunomodulatory composition was furtherprocessed via heat-inactivation, i.e. the suspension was boiled on a hotplate for 5 minutes. The evaporated water was then replaced (with milliQwater) to make the suspension up to the initial volume. This was thendiluted and plated as described above.

The plates were incubated away from light at 30° C. for 18 hours andthen another 24 hours at room temperature (22° C.).

Results

After the incubation period, no visible colonies were observed on anyplate containing filtered or heat-inactivated immunomodulatorycompositions. Similarly, no colonies grew on the cetrimide agar platedwith sieved immunomodulatory composition dilutions B-E. On the platecontaining the dilution A of the sieved immunomodulatory compositionthere were few white colonies, but their colour and appearance were notthose of Pseudomonas aeruginosa colonies but colonies of another speciesof Pseudomonas.

The technical effect is that the immunomodulatory compositions are freeof Pseudomonas aeruginosa. Hence, the immunomodulatory compositions canbe used without a risk of Pseudomonas aeruginosa infection.

Example 7: Potential Bacterial Pathogens in Illumina Sequencing Methods

Sampling, Sample Preparation and Sequence Processing

Here we compared freeze dried immunomodulatory composition (3 samples)to mineral soil samples from soil aggregate producer (5 samples), tomineral soils from daycare yards (6 samples) and to natural forestsamples (2 samples). Samples from soil aggregate producer, from daycareyard and from natural forest soil represent a normal environment thaturban people encounter in their everyday life.

Mineral soil samples were collected from playgrounds of 3 daycarecenters in the city center in Lahti, Finland. Altogether 6 samples fromyards of the daycare centers were collected. Surface soil (depth 1-2 cm)was collected. At each of the three daycare yards, the first sample wastaken from a sandbox. The second sample was taken from other site withmineral soil, such as under a swing set. For each sample, three ca. 2 gsubsamples were collected, pooled and mixed thoroughly. Distance betweensubsampling points was at least 30 cm.

The sampling of other sample types, sample preparation and sequenceprocessing of these samples has been described in example 4. However,here we used genus level data i.e. the OTUs were assigned to genera.During the sequence processing in Mothur, the sequences were classifiedusing the Mothur version of Bayesian classifier (Wang et al. 2007) withthe RDP training set version 9 (Cole et al 2009) and a consensustaxonomy for each OTU was generated. Then, the number of sequences ineach OTU that had been classified into same genus were summed.Additionally, we used data that was processed in three different ways.First, we used data that was subsampled (rarefied) to 2000 sequences aswas also used in example 4. Three mineral soil samples from soilaggregate producer had less than 2000 sequences and thus were notincluded in this comparison, but these samples were included in theother two data variants. Second, we used this same data withoutsubsampling. And third, we used the unsubsampled data and converted itto relative abundances (i.e. number of sequences was divided with totalnumber of sequences in a sample and multiplied with 100 to providepercentage frequency of each genus in each sample). Unsubsampled datawas used because subsampling can remove some rare OTUs belonging topotentially pathogenic genera.

Data Processing

We first inspected the minimum, maximum, mean and standard deviation ineach of the four groups of samples for nine genera that includepathogenic species. These were Acinetobacter, Bacillus, Lactobacillus,Mycobacterium, Neisseria, Nocardia, Pseudomonas, Sphingomonas andStreptococcus.

Then, we picked all the genera that have been listed to includepotentially pathogenic species in Taylor et al. (2001) appendix A. Wecounted the abundance i.e. the sum of the number of sequences in eachgenera. Then we counted the minimum, maximum, mean and standarddeviation in each of the four groups of samples.

Results

The mean and maximum of the abundance of each of the selected genera wasalways lower or equal in freeze dried immunomodulatory compositioncompared to the three other sample groups that represent the normalenvironment that urban people encounter in their everyday life (Tables6-8). The abundance of potentially pathogenic genera, measured as 16Scopies, was low in the immunomodulatory composition no matter if thedata was subsampled (Table 6), non-subsampled (Table 7) or relativeabundance data was compared (Table 8). This supports the view that theimmunomodulatory composition is safer to handle than mineral soil atdaycare yards, and safer or equally safe as commercial mineral soilproducts.

Altogether 34 genera of all the 136 genera listed in Taylor et al.(2001) were found in the whole data. Mineral soil samples from daycareyard had the highest number (i.e. 23) of potentially pathogenic genera(Table 9). Also samples from soil aggregate producer included more (i.e.19) genera than freeze dried immunomodulatory composition and naturalforest soil which had 15 and 14 genera, respectively. None of the generawere unique to freeze dried immunomodulatory composition meaning that ifa genus was found from freeze dried immunomodulatory composition, it wasalso found from one of the reference samples that represent the normalenvironment that urban people encounter in their everyday life.

When all the sequences that belong to the potentially pathogenic generalisted in Taylor et al. (2001) were summed, the mean and maximum valueswere lower in the freeze dried immunomodulatory composition than mineralsoil at daycare yards and this result was robust for data managementtype (Table 9). Also when considering relative abundances, mineral soilssamples from aggregate producer had higher amount of sequences belongingto potentially pathogenic genera. Noteworthily, as evidenced in Table 9,the between-sample variability is virtually missing in the total numberof potentially pathogenic bacterial 16 S sequences.

As seen in Tables 6-8, the abundance and relative abundance ofLactobacillus, Mycobacterium and Acinetobacter are low in theimmunomodulatory composition. This means that the beneficial strainsisolated in those genera cannot be the reason for the strongimmunoregulatory response evidenced in the example 3. The technicaleffect is that the strong immunoregulatory response is caused by theimmunomodulatory composition comprising non-culturable microbialcommunity as described in the current invention.

The technical effects of the findings are that the immunomodulatorycompositions according to the present invention are safe to use,partially because their quality is homogeneous. Importantly, theimmunomodulatory composition did not contain any potentially pathogenicgenera that were not present in everyday urban living environment.Because the richness and diversity of non-pathogenic genera areexceptionally high in the immunnomodulatory composition (Example 4),another technical effect is that the immunomodulatory compositionsaccording to the present invention expose urban subjects to richmicrobial community without an additional risk of infections. Thisdecreases the abundance of immune mediated disorders.

TABLE 6 Number of sequences of potentially pathogenic genera detected insamples of freeze dried immunomodulatory composition, mineral soils fromaggregate producers, surface mineral soils from daycare yards andnatural forest soils. Subsampling was done to 2000 sequences. Data:adjusted for control, subsampled to 2000 Acinetobacter BacillusLactobacillus Mycobacterium Neisseria Nocardia Pseudomonas SphingomonasStreptococcus Freeze dried immunomodulatory composition (n = 3) Min 2 00 1 0 0 1 14 0 Max 4 0 1 2 0 0 11 19 0 Mean 3 0 0 1 0 0 6 17 0 St Dev 10 1 1 0 0 5 3 0 Mineral soil-aggregate producer (n = 2) Min 1 0 0 0 0 07 7 0 Max 2 0 1 1 0 0 16 9 2 Mean 2 0 1 1 0 0 12 8 1 St Dev 1 0 1 1 0 06 1 1 Mineral soil-daycare yard (n = 6) Min 2 0 0 5 0 0 3 50 0 Max 9 0 115 0 0 28 125 0 Mean 4 0 0 10 0 0 16 83 0 St Dev 3 0 1 4 0 0 10 28 0Natural forest soil (n = 2) Min 0 0 0 3 0 0 0 0 0 Max 6 0 0 33 0 1 6 1 0Mean 3 0 0 18 0 1 3 1 0 St Dev 4 0 0 21 0 1 4 1 0

TABLE 7 Number of sequences of potentially pathogenic genera detected insamples of freeze dried immunomodulatory composition, mineral soils fromaggregate producers, surface mineral soils from daycare yards andnatural forest soils. No subsampling. Data: adjusted for control,unsubsampled Acinetobacter Bacillus Lactobacillus MycobacteriumNeisseria Nocardia Pseudomonas Sphingomonas Streptococcus Freeze driedimmunomodulatory composition (n = 3) Min 5 0 0 12 0 0 3 99 0 Max 19 0 215 0 0 7 134 1 Mean 13 0 1 14 0 0 5 111 0 St Dev 7 0 1 2 0 0 2 20 1Mineral soil-aggregate producer (n = 5) Min 0 0 0 0 0 0 0 5 0 Max 9 0 35 0 0 22 192 10 Mean 3 0 1 1 0 0 6 55 2 St Dev 4 0 1 2 0 0 9 78 4Mineral soil-daycare yard (n = 6) Min 5 0 0 38 0 0 9 363 0 Max 56 0 1 921 1 56 969 1 Mean 27 0 1 60 0 0 31 594 0 St Dev 20 0 0 23 0 0 21 230 1Natural forest soil (n = 2) Min 0 0 0 38 0 0 0 0 0 Max 21 0 0 249 0 15 01 0 Mean 11 0 0 144 0 8 0 1 0 St Dev 15 0 0 149 0 11 0 1 0

TABLE 8 Relative number of sequences of potentially pathogenic generadetected in samples of freeze dried immunomodulatory composition,mineral soils from aggregate producers, surface mineral soils fromdaycare yards and natural forest soils. No subsampling. Data: adjustedfor control, relative abundances Acinetobacter Bacillus LactobacillusMycobacterium Neisseria Nocardia Pseudomonas Sphingomonas StreptococcusFreeze dried immunomodulatory composition (n = 3) Min 0.0 0.0 0.0 0.10.0 0.0 0.1 0.7 0.0 Max 0.1 0.0 0.0 0.1 0.0 0.0 0.4 1.0 0.0 Mean 0.1 0.00.0 0.1 0.0 0.0 0.3 0.8 0.0 St Dev 0.1 0.0 0.0 0.0 0.0 0.0 0.1 0.2 0.0Mineral soil-aggregate producer (n = 5) Min 0.0 0.0 0.0 0.0 0.0 0.0 0.00.3 0.0 Max 0.1 0.0 0.3 0.0 0.0 0.0 0.6 9.7 0.1 Mean 0.0 0.0 0.1 0.0 0.00.0 0.2 2.7 0.0 St Dev 0.0 0.0 0.1 0.0 0.0 0.0 0.3 4.0 0.0 Mineralsoil-daycare yard (n = 6) Min 0.0 0.0 0.0 0.3 0.0 0.0 0.1 3.1 0.0 Max0.4 0.0 0.0 0.6 0.0 0.0 1.3 6.5 0.0 Mean 0.2 0.0 0.0 0.4 0.0 0.0 0.7 4.20.0 St Dev 0.1 0.0 0.0 0.1 0.0 0.0 0.5 1.3 0.0 Natural forest soil (n =2) Min 0.0 0.0 0.0 0.3 0.0 0.0 0.0 0.0 0.0 Max 0.2 0.0 0.0 1.7 0.0 0.10.4 0.0 0.0 Mean 0.1 0.0 0.0 1.0 0.0 0.1 0.2 0.0 0.0 St Dev 0.1 0.0 0.01.0 0.0 0.1 0.3 0.0 0.0

TABLE 9 Sum of sequences of potentially pathogenic bacteria detected insamples of freeze dried immunomodulatory composition, mineral soils fromaggregate producers, surface mineral soils from daycare yards andnatural forest soils. Results based on unsubsampled and subsampled dataas well as relative abundance data 5 are shown. Also the number ofpotentially pathogenic genera is shown. Number of Number of sequences inpotentially potentially pathogenic pathogenic genera genera Data:adjusted for control, unsubsampled Mean Min Max StdDev Freeze dried 317303 341 21 15 immunomodulatory composition Mineral soil - aggregate 1157 217 103 19 producer Mineral soil - daycare yard 974 574 1350 281 23Natural forest soil 223 166 279 80 14 Data: adjusted for control,subsampled to 2000 Mean Min Max StdDev Freeze dried 47 36 56 10immunomodulatory composition Mineral soil - aggregate 30 30 30 0producer Mineral soil - daycare yard 141 108 175 29 Natural forest soil32 24 40 11 Data: adjusted for control, relative abundances Mean Min MaxStdDev Freeze dried 2.4 2.3 2.4 0 immunomodulatory composition Mineralsoil - aggregate 4.0 1.3 10.9 4 producer Mineral soil - daycare yard 6.95.0 9.0 1 Natural forest soil 1.6 1.2 1.9 1

Example 8—Baby and Toddler Product Descriptions

Packets of Immunomodulatory Material

Examples of the immunomodulatory compositions that can be used hereinare dried and sieved moss (particle size less than 1 mm), sievedcomposition used in example 2 and freeze dried and inactivatedimmunomodulatory composition used in example 3. These materials aresuitable for modifying skin microbial community as their microbialabundance, diversity and richness are high, and they do not containsharp particles. An example of high microbial abundance of theimmunomodulatory compositions is given in FIG. 6.

The packets containing immunomodulatory composition are made of agossamer-like fabric. A layer of immunomodulatory composition is placedinside the packets, and they come in two sizes: 15×21 cm (A5) and 10×10cm. The smaller packet has been tested for choking hazard with a testcylinder (small objects choke tester) provided by Tukes (Finnish Safetyand Chemical Agency). According to the test, the packets do not pose achoking hazard. The smaller immunomodulatory packets are used as part ofinfant hats and security blankets. The bigger immunomodulatory packetsare used with the other products. The thin permeable fabric of thepackets allows the immunomodulatory components of the material passthrough on the skin of the user. Thus, the microbial community on theskin changes as evidenced in example 9.

Infant Hat

The infant hat is made of cotton jersey, and inside its front (coveringthe forehead) there is a pocket for the immunomodulatory packet (10×10cm). The size of the hat is 60-86 cm (3 months to 3 years), and it canbe adjusted by tying the top end of the hat with a ribbon, hair elastic,or equivalent.

The immunomodulatory packet that is placed inside the hat has beentested for choking hazard with a choke tube tester by Tukes, and as aresult no choking hazard exists. The immunomodulatory components ofmaterial are able to pass through the layers of fabric and thus act onthe skin of the forehead as evidenced in example 9.

Infant hat is illustrated in FIGS. 7 and 8. Technical effect isdescribed in FIG. 7.

Building Blocks

Another example of an article is in the form of foam building blockscovered with cotton fabric, and they are 7×37×50 cm in size. They areillustrated and the technical effect is described in FIG. 9. Childrencan safely climb on the blocks, lift up the blocks, and build with theblocks. These activities enable the immunomodulatory components of theimmunomodulatory material to scatter on and around the children. Thiswill in turn activate immunoregulatory mechanisms i.e. a technicaleffect. This technical effect can partially also be produced by inhalingthe immunomodulatory components while using the product.

The fabric cover has a zipper, and four immunomodulatory packets (15×21cm) can be placed inside the cover. The packets can be changed regularlyor when advised.

The packets can only be removed by undoing the zipper on the cover. Thecover is made of 100% cotton fabric. The cotton fabric used here hasbeen certified according to Öko-Tex 100 standard in the product class I(the highest level of product class), but this certification is notnecessary for the desired effect. The other fabric parameters are: 100%cotton, plain weave, average yarn number of 18.33, decitex measurementper single yarn is 500. The chemicals and dyes used for the making ofthe fabric are in accordance with the Reach regulation of the EuropeanUnion.

Infant Security Blanket

Another example of an article is an infant security blanket illustratedin FIGS. 10 and FIG. 11. It is made of gauze-like cotton fabric, and itfulfills the above mentioned security standards. It is a square piece offabric that is 50×50 cm in size. A 30 cm long cotton ribbon is sewn nearthe middle of the square, and the attachment point of the ribbon dividesthe ribbon into two equal lengths (=15 cm and 15 cm). This length waschosen because it will not pose a choking hazard. The piece of fabric isthen folded in half, and the immunomodulatory packet (10×10 cm) isinserted in the middle and secured in place by tying the ends of theribbon around the fabric.

The standards for safety of children's clothing or toys do not apply toinfant security blankets. General provisions, however, require generalconsumer goods to be safe. The immunomodulatory packet placed inside thesecurity blanket has been tested with the small objects choke testerprovided by Tukes to exclude any choking hazard.

Pillow Case and Duvet Cover

The pillow case and the duvet cover are sized for children: the pillowcase is 40×50 cm whereas the duvet cover is 80×115 cm. There are two andfour pockets for the immunomodulatory packets inside the pillow case andthe duvet cover, respectively. The pockets are sewn inside the covers,and they are A5 (15×21 cm) in size. The pockets are located on bothsides of the inserted pillow or blanket, and they need to accessed frominside to remove the immunomodulatory packets as the packets are lockedin place by the pocket folds. The pockets are as inconspicuous aspossible for the user and should not cause any lumps or bulges on thesurface of a pillow or a blanket. The pillow case and duvet cover areillustrated and their technical effect is described in FIG. 12.

Example 9. Skin Bacterial Composition Before and After Use ofImmunomodulatory Composition Methods

Testing the Packets and Sampling

The immunomodulatory composition used herein was dried, crushed andmixed Sphagnum moss (particle size less than 1 mm). A layer ofimmunomodulatory composition was placed inside a fabric packet of size10×10 cm. Three different types of fabrics were tested: airlaid materialST047DIA (thickness 0.44 mm), airlaid material DS100 (thickness 0.85 mm)and cotton fabric. Airlaid materials are used for manufacturingdifferent kind of products such as table tops, napkins, and laboratorypads. Cotton fabric was the same material which was used in baby andtoddler products described in example 8.

Two volunteers conducted the experiment. Both volunteers tested onepacket made of ST047DIA and one packet made either of DS100 or cottonfabric. The packets were placed on the inner forearms of the volunteers.Packets were tied on with a clean disposable self-adhesive bandage. Thevolunteers were exposed to the packets for 3 hours and 45 minutes. Skinswabs (5×5 cm area, 10 seconds) were taken just before placing thepackets and immediately after they were removed. A stick with a cottonwool tip was first wetted in Tween® 20, used in sampling and placed intoa sterile polyethylene sample tube.

Bacterial composition on skin before and after use of the packets wascompared to the bacterial composition in the Spagnum moss that was usedas a raw material for the packets. The preparation of the moss sample isdescribed in example 4.

Sample Preparation to MiSeq Sequencing for Skin Swab Samples

Skin swab samples were stored in deep freezer (<−70° C.) in tubescontaining Tween 20 (MP Biomedicals) (0.1%)+NaCl (0.1 M, J. T.Baker)before DNA extraction. Total DNA was extracted from samples usingPowerSoil® DNA Isolation Kit (MoBio Laboratories, Inc., Carlsbad,Calif., USA) according to the manufacturer's standard protocol. The swabwas transferred to the PowerBead tube for a homogenization and lysisprocedure. DNA was checked with agarose gel (1.5%) electrophoresis (120V, 30 min). Total DNA concentration was measured with Quant-iT™PicoGreen® dsDNA reagent kit (Thermo scientific, MA, USA).

The V4 region within the 16S ribosomal RNA (rRNA) gene was amplified byprimary PCR as triplicates using 505F and 806R primers (Caporaso et al.2012). Primary PCR was carried out in a reaction mixture (reactionvolume 50 μl) consisting of 1 μl each of 10 mM deoxynucleosidetriphosphates (dNTPs; Thermo scientific, MA, USA) 5 μl forward primer505F (10 μM; 5′-GTGCCAGCMGCCGCGGTAA-3′) and 5 μl reverse primer 806R (10μM; 5′-GGACTACHVGGGTWTCTAAT-3′), 0.5 μl 2 U/μl Phusion Green Hot StartII High-Fidelity DNA polymerase (Thermo scientific, MA, USA), 10 μl 5×Green HF PCR buffer (F-537), 5 μl template DNA and 23.5 μl sterilewater. The PCR reaction was run in a thermocycler (MJ Research, MA, USA)as follows: initial denaturation at 98° C. for 5 min, followed by 30cycles with denaturation at 94° C. for 1 min, annealing for 10 sec at50° C. and extension for 1 min at 72° C., and then a final extension at72° C. for 10 min. A positive control (Cupriavidus necator JMP134, DSM4058) was included in PRC runs to ensure that the PCR worked, and anegative control (sterile water) was run to detect any possiblecontamination. DNA was detected with agarose gel (1.5%) electrophoresis(120 V, 1 h). The PCR products were purified using Agencourt AMPure XPsolution (Beckman Coulter Ins.) to reduce carryover of primary PCRprimers. Triplicates of the cleaned amplicons were pooled and diluted1:5.

Cleaned and diluted primary PCR products were targeted in the secondaryPCR (TagPCR). Reaction mixture to the TagPCR was equal as above exceptreverse primer included a 12 bp unique Multiplexing Identifier tag(MID-806R). Amplification program was the same as above except therewere only seven cycles for soil products and ten cycles for othersamples. TagPCR products were detected on agarose gel (1.5%)electrophoresis (120 V, 1 h), purified with Agencourt AMPure, pooled andthe DNA concentration was measured with PicoGreen. The sequencing wasperformed at the Kansas State University using Illumina MiSeq platform.The sequencing was performed using Illumina MiSeq platform with a 2×300bp version 3 kit sequencing kit according to manufacturer's protocol.The GeneRead DNA Library I Core Kit (Qiagen, catalog #180432) was usedto ligate IIlumina's TruSeq adapters to amplicons.

Sequence Processing

Sequence data for skin swab samples and the moss sample were analyzedtogether using Mothur-program (version v.1.38.1; Schloss et al. 2009).The sequence processing protocol followed the same protocol described inexample 4, with the exception that samples were processed unrarefieduntil the end of the protocol. For each OTU the number of sequencesfound in negative controls (extraction negative, per negative) weresubtracted from each sample. The final OTU-table was rarefied to 2000similarly as in example 4.

Data Analyses

Number of OTUs, Shannon diversity and Fisher's alpha were calculatedusing functions diversity, fisher.alpha and specnumber in R packagevegan. Principal Coordinate Analysis (Gower 1966) was performed usingcmdscale function in R package stats.

Results

Bacterial richness, Shannon diversity index and Fisher alpha were alwayshigher in samples taken after the use of packets containingimmunomodulatory composition compared to the samples taken before theuse, see Table 10. The bacterial community composition in skin swabsamples was different, depending on whether the immunomodulatorycomposition was used or not, see FIG. 13. The technical effect is thatthe use of the immunomodulatory composition changes skin microbialcommunity, e.g. it increases Shannon diversity index and bacterialrichness.

TABLE 10 Bacterial richness (i.e. number of OTUs), Shannon diversityindex i.e. Shannon index and Fisher alpha of skin swab samples takenbefore and after the use of packets filled with an immunomodulatorycomposition. Also the values for Spagnum moss used as raw material tomanufacture the immunomodulatory composition are shown. Bag type SampleNumber of OTUs Shannon index Fisher alpha DS100 Before 123 2.74 28.94DS100 After 159 3.27 40.59 Cotton fabric Before 85 2.62 18.01 Cottonfabric After 142 3.59 34.93 ST047DIA_1 Before 109 2.41 24.75 ST047DIA_1After 141 2.87 34.61 ST047DIA_2 Before 103 2.90 23.01 ST047DIA_2 After136 3.27 33.00 Moss 194 3.46 53.07

Example 10. Particle Size in the Sieved Immunomodulatory Composition

Sieved immunomodulatory composition was manufactured as described inexample 2, e.g. ∅5 mm and ∅2 mm sieves were used. To find out particlesizes of the remaining particles, 50 g of soil mixture was driedovernight (˜18hrs) at 70° C. Weight after drying was 26.48 g. This wassieved through 5 sieves with Retsch AS 200 analytical sieve shaker withamplitude of 50 s-1 for 10 minutes. The results show that >50% orparticles were smaller than ∅ (Table 11). Together with the results ofthe previous examples, the technical effect is that sieving is suitablefor manufacturing immunomodulatory compositions that are safe andeffective.

TABLE 11 Aperture size of particles in the sieved immunomodulatorymixture. aperture size sieve weight total weight after soil on (micron)(g) sieving (g) sieve (g) % 1000 304.32 316.57 12.25 46.3 500 305.5311.43 5.93 22.4 250 281.82 286.06 4.24 16.0 125 263.79 265.89 2.1 7.963 251.4 252.42 1.02 3.9 collecting pan 353.66 354.6 0.94 3.5

Example 11

The experimental setup of example 2 was followed. In short, sevenhealthy adults that live in urban conditions were in control group thatcontinued normal life. At the same time, 7 healthy adults that live inurban conditions were in an exposure group that “washed” their handswith an immunomodulatory preparation 3 times a day for two weeks.Otherwise also the exposure group lived normal life. Theimmunomodulatory preparation is described in example 2. Stool microbiotawas sampled and analyzed as in experiment 2, in the beginning and in theend of the experiment. Skin microbiota was sampled and analyzedsimilarly to example 1, in the beginning and in the end of theexperiment. The skin microbial sample was always taken from the forearm,while only palms and fingers were exposed to the immunomodulatorymaterial.

We compared the change in microbial diversity in the exposure group tothe expression of an immunoregulatory cytokines (TGF-beta) in peripheralblood mononuclear cells (PBMCs) at the end of the exposure period. Thechange in both in skin and stool microbial diversity was associated withTGF-beta expression (R2=0.971, p=0.001).

The results show that the immunomodulatory material described in thisinvention causes an immunomodulatory effect according to the currentinvention.

Without limiting the scope and interpretation of the patent claims,certain technical effects of one or more of the aspects embodimentsdisclosed herein are listed in the following: A technical effect isincreased microbial diversity on or in a human or animal subject. Atechnical effect is increased microbial richness on or in a human oranimal subject. A technical effect is increased microbial abundance onor in a human or animal subject. A technical effect is induced IL-10expression on or in a human or animal subject. A technical effect isaltered IFN-gamma expression on or in a human or animal subject. Thesefive technical effects are shown in examples 1-3.

A technical effect of wearing or playing or otherwise being in contactwith an immunomodulatory product is increased microbial diversity,richness and/or abundance as evidenced in examples 8-9 and 1-3.

A technical effect of immunomodulatory compositions obtainable byprocessing material derived from nature is more comfortable texture andfeel of the immunomodulatory composition; natural materials,particularly soil and plant parts, have particles with sharp edges thatcan hurt. These sharp edged particles are removed in e.g. sieving, as inexamples 2 and 10.

A technical effect of modification of material derived from nature ishigh diversity, richness and abundance or non-culturable microbes inimmunomodulatory compositions, as evidenced in example 4.

A technical effect of freeze drying or sterilization is reduced ormissing activity of pathogens. The modifications, however, stillfacilitate the immunostimulation by immunomodulatory compositions, asevidenced in example 3.

A technical effect of sieving and many other modifications describedabove is reduced heterogeneity of material derived from nature, as shownin example 7. In heterogeneous materials, patches of single microbialstrains are likely, which allows high abundance of pathogenic OTUs.Another technical effect is absence or low abundance of pathogenicmicrobes, as evidence in examples 5-7. Pathogenic microbes in naturalmaterials, such as soil, are the reason why soil and other naturalproducts are not safe be used as such to modulate immune system.

A technical effect of several modifications, e.g. sieving and filtering,is non-existing risk of multicellular parasites such as lice and ticks,because these are too large to pass sieving, filtering, or unable tosurvive in sterilization or evaporation.

The foregoing description has provided by way of non-limiting examplesof particular implementations and embodiments of the invention a fulland informative description of the best mode presently contemplated bythe inventors for carrying out the invention. It is however clear to aperson skilled in the art that the invention is not restricted todetails of the embodiments presented above, but that it can beimplemented in other embodiments using equivalent means withoutdeviating from the characteristics of the invention.

Furthermore, some of the features of the above-disclosed embodiments ofthis invention may be used to advantage without the corresponding use ofother features. As such, the foregoing description should be consideredas merely illustrative of the principles of the present invention, andnot in limitation thereof. Hence, the scope of the invention is onlyrestricted by the appended patent claims.

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1. A food ingredient comprising a microbial community of non-culturablebacteria, wherein the microbial diversity is at least 3 at Shannondiversity index; the microbial richness is at least 130 operationaltaxonomic units; the microbial abundance is at least 1,000,000 bacterial16S copies g-¹ ww; and the abundance of pathogens is below 500 16S genesequences per 0.25 g sample in bacterial genera Acinetobacter,Actinomyces, Aerococcus, Aeromonas, Arcobacter, Bacillus, Bacteroides,Bifidobacterium, Brevibacillus, Brevundimonas, Chryseobacterium,Corynebacterium, Fibrobacter, Finegoldia, Gemella, Lactobacillus,Legionella, Leptotrichia, Moraxella, Mycobacterium, Myroides, Neisseria,Nocardia, Paenibacillus, Prevotella, Pseudomonas, Pseudonocardia,Psychrobacter, Rhodococcus, Rickettsia, Saccharomonospora, Sphingomonas,Stenotrophomonas, Streptococcus, and Treponema.
 2. The food ingredientof claim 1 further comprising at least one additive selected from apreservative, a coloring agent, a bulking agent, and a stabilizer. 3.The food ingredient of claim 1, in which the food ingredient is in aform of a dose unit.
 4. The food ingredient of claim 1, in which thefood ingredient is in a form of a pill or a capsule.
 5. The foodingredient of claim 1, in which the microbial community comprises viablenon-culturable bacteria.
 6. The food ingredient of claim 1, in which themicrobial community comprises inactivated microbes and pathogens.
 7. Thefood ingredient of claim 1, in which the non-cultural bacteria arehomogeneously distributed.
 8. A method for maintaining and strengtheningimmune system of a subject comprising administering to the subject aneffective amount of the food ingredient according to claim
 1. 9. Themethod of claim 8, in which the administering is for at least 2 weeksand carried out sequentially at intervals of not more than 7 days. 10.The method according to claim 8, in which the administering is carriedout daily.